Isolation and characterization of the csa operon (ETEC-CS4 pili) and methods of using same

ABSTRACT

Compositions comprising products of the csa operon, an isolated nucleic acid encoding the csa operon or functional fragments thereof, purified polypeptide products of the csa operon or functional fragments thereof, methods of eliciting an immune response to these products, and methods of producing products of the csa operon are disclosed herein.

Related Applications

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/198,626, filed Apr. 20, 2000, which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The disclosed invention relates to the isolation andcharacterization of the csa operon, which encodes the CS4 pili and itsuse as an immunogenic agent with utility in preventing ETEC colonizationof a subject.

[0004] 2. Description of the Related Art

[0005] Human ETEC strains are a major cause of diarrhea in infants andyoung children in developing countries (Black et al., Lancet, I: 141-143(1981); Levine, J. Infect. Dis., 155:377-389 (1987); Qadri, et al., J.Clin. Microbiol., 38:27-31 (2000)), which account for a high rate ofinfantile morbidity and mortality. Human ETEC strains are also a majorcause of travelers' diarrhea. (Black, Rev. Infect. Dis., 8S:S131-S135(1986); DuPont et al., N. Engl. J. Med., 285:1520-1521 (1976); Hyams etal., N. Engl. J. Med., 325:1423-1428 (1991); Merson, et al., N. Engl. J.Med., 294:1299-1305 (1976)). ETEC infection is characterized by waterydiarrhea often accompanied by low-grade fever, abdominal cramps, malaiseand vomiting.

[0006] ETEC strains colonize the small bowel lumen by means of surfacepili called colonization factor antigens (CFA), and coli surfaceantigens (CS), and cause diarrhea through the action of heat labile (LT)and/or heat stable (ST) enterotoxins. ETEC fimbriae are proteinaceousfilaments exhibiting different morphologies such as rigid rod likeshapes of 2-7 nm in diameter, fibrilar thin flexible wiry structures, orbundles. (Gaastra et al., Trends. Microbiol., 4:444-452 (1996)).

[0007] Human ETEC strains display a variety of over 20 serologicallydistinct pili on their cell surfaces. The most common human ETEC strainsexpress CFA/I, CFA/II and CFA/IV (Levine, et al., “Fimbrial vaccines,”In P. Klemm (ed.), Fimbriae: adhesion biogenics, genetics and vaccines,Boca Raton: CRC Press, 1994; McConnell, et al., Epidemiol. Infect., 106:477-484 (1991)). CFA/I produces a single type of fimbriae, while CFA/IIand CFA/IV strains produce several types of coli surface antigens.CFA/II strains express CS1, CS2 and CS3; and CFA/IV strains (originallycalled PCF8775) express the nonpilus antigen CS6 either alone ortogether with CS4 or CS5 fimbria. (McConnell, et al., Infect. Immun.,56:1974-1980 (1988); McConnell, et al., FEMS Microbiol. Lett.,52:105-108 (1989); Svennerholm, et al., Infect. Immun., 56:523-528(1988); Thomas, et al., J. Gen. Microbiol., 131:2319-2326 (1985)). Theoccurrence of CS4⁺ CS6⁺ producing strains is restricted to serotypeO25:H42. (McConnell, et al., Infect. Immun., 56:1974-1980 (1988);Willshaw, et al., FEMS Microbiol. Lett., 49:473-478 (1988); Willshaw, etal., FEMS Microbiol. Lett., 56:255-260 (1990); Willshaw, et al., FEMSMicrobiol. Lett., 66:125-129 (1991)).

[0008] The CS4 pili is rigid, 7 nm in diameter, and is composed ofsubunits with a molecular mass of 17.0 kDa. (Knutton, et al., Infect.Immun., 57:3364-3371 (1989); McConnell, et al., Infect. Immun.,56:1974-1980 (1988); Wolf, et al., Infect. Immun., 57:164-173 (1989)).Because of their epidemiological importance and due to the fact thatcross protection does not occur between strains of ETEC expressingdifferent fimbriae, at least these CFA/I and CS1-CS6 fimbrial types mustbe included in a broad spectrum ETEC vaccine, (Gaastra et al., Trends.Microbiol., 4:444-452 (1996); Levine, J. Pediatr. Gastroenterol. Nutr.,In Press (2000); Levine, et al., “Fimbrial vaccines,” In P. Klemm (ed.),Fimbriae: adhesion, biogenics, genetics and vaccines, Boca Raton: CRCPress, 1994). Of these seven important fimbriae, only the genes encodingCS4 have not been cloned and sequenced.

[0009] The genes that are required for the expression of functional piliare characteristically linked in gene clusters (Sakellaris and Scott,Mol. Microbiol., 30:681-687 (1998)), and consist of the structuralgenes, assembly cassette genes and regulatory genes. The assemblycassette genes include chaperone and usher genes. The chaperone proteinis thought to bind to fimbrial subunit proteins in the periplasmic spaceand prevent premature folding and degradation. The usher proteins areouter membrane proteins that serve as pores for the transport andassembly of the fimbriae. The structural gene encodes for the pilinprotein that forms the fimbriae that is composed of repeated subunits ofthe pilin protein. Some fimbriae such as, CFA/I, CS1 and CS2, contain aminor pilin protein which is associated with the pili tip, that isprobably involved in the attachment of the bacteria to the cellreceptors. (Sakellaris, et al., Proc. Natl. Acad. Sci. U.S.A.,96:12828-12832 (1999)). Fimbria expression is controlled by genes suchas rns and cfaD that are similar to the araC family of transcriptionalregulators that positively regulate transcription. (Grewal et al.,Vaccine, 11:221-226 (1993); de Haan et al., FEMS Microbiol. Lett.,67:341-346 (1991); 19, 21, Savelkoul, et al., Microb. Pathog., 8:91-99(1990)).

[0010] Genes encoding ETEC fimbriae have been located on large plasmids[CFA/I, (Hamers et al., Microb. Pathog., 6:297-309 (1989); 20); CS1,(Froehlich et al., Mol. Microbiol., 12:387-401 (1994)); CS3,(Jalajakumari, et al., Mol Microbiol., 3:1685-1695 (1989), Manning, etal., Mol. Gen. Genet., 200:322-327 (1985)); CS5, (Duthy, et al., J.Bacteriol., 181:5847-5851 (1999)) and CS6, (Wolf, et al., FEMSMicrobiol. Lett., 148:35-42 (1997))], or on the chromosome [CS2,(Froehlich et al., Mol. Microbiol., 12:387-401 (1994), Froehlich et al.,Infect. Immun., 63:4849-4856 (1995))]. Early experiments to locate theCS4 encoding genes revealed disparate results without conclusivelocalization, (Sommerfelt, et al., Microb. Pathog., 11:297-304 (1991);Sommerfelt, et al., Infect. Immun., 60:3799-3806 (1992); Sommerfelt, etal., J. Clin. Microbiol., 30:1823-1828 (1992); Willshaw, et al., FEMSMicrobiol. Lett., 56:255-260 (1990); Wolf, et al., Infect. Immun.,57:164-173 (1989)).

[0011] Because the attachment of ETEC strains to intestinal cells iscrucial for establishment of infection, the prevention of disease isbased mainly upon immune responses against the pili that interfere withthe attachment process. Studies performed with 14 healthy humanvolunteers who ingested 5×10⁸ E. coli E24377A (0139:H28), a CS1 and CS3producing strain, showed that all 14 became colonized, 9 developed atypical diarrheal syndrome, and 6 of these ill persons manifested asignificant increase in serum IgG antibody to purified CS1 and CS3antigens. Levine, M. M., et al., Infect. Immun., 44:409-420 (1984).These results suggest that the fimbria play a role in pathogenesis, andstimulate an immune response.

[0012] Passive protection against ETEC infection was demonstrated in aclinical trial done at the University of Maryland, which demonstratedthat oral prophylaxis with hyperimmune anti CFA/I immunoglobulinprovided 90% protection against diarrhea caused by oral challenge with10⁹ cfu of ETEC strain H10407, a CFA/I producing strain. Tacket, et al.,N. Eng. J. Med., 318: 1240 (1988). In another study oral immunizationwith purified CS1 and CS3 antigens encapsulated in biodegradable polymermicrospheres were used to induce the development of IgA anti CS ASC(Antibody Secreting Cells) and jejunal fluid secretory IgA anti CS in50% of the vaccinees. Levine, et al., Fimbriae (pili) adhesions asvaccines, in Protein-Carbohydrate Interactions in Biological Systems.The molecular Biology of Microbial Pathogenicity, Lark, et al., Eds.,Academic Press, London, p 154, 1986. This exposure also protected 30% ofvaccinees from diarrhea following challenge with the virulent ETECE24377A (CS1⁺CS3⁺LT⁺ST⁺) strain. A protective efficacy of 75% wasdemonstrated by immunization with the attenuated ETEC strain. Feedingvolunteers with 5×10¹⁰ live E. coli E1392-75-2A (O6:H16) a CS1⁺ CS3⁺ LT⁻ST⁻ strain, induced significant rise in intestinal fluid secretory IgAantibodies to CS1 and CS3 fimbria, and conferred protection to 9/12volunteers that were challenged with a virulent heterologous serotypestrain ETEC E24377A (O139:H28).

[0013] Another approach to develop an ETEC vaccine is to immunize withkilled mixed ETEC strains. This type of vaccine is based on the factthat prior infection with an ETEC strain elicits protective immunityagainst a clinical illness that might be caused from subsequent exposureto the homologous strain. Oral immunization of children and adultvolunteers with such a vaccine resulted in significant intestinal IgAresponses against the CFA and CS components of the various strains. Thevaccine induced high level of intestinal IgA antibody, IgA antibody ASCin the blood, and serum antibodies towards the colonization factorantigens. Jertbom, et al., Vaccine 16:255 (1997). Those results indicatethat antibodies towards ETEC pilis provide protection against diarrheacaused by ETEC strains.

[0014] A combined vaccine against diarrheal disease caused by Shigella,Salmonella and ETEC has been proposed. These vaccines are composed oflive attenuated Shigella or Salmonella strains that express ETECfimbriae. The recombinant bacteria colonize the intestine and inducedthe mucosal and systemic immune response against the bacteria and thepili. Noriega et al., (Infect. Immun., 64:23-27 (1996)) co-expressedCFA/I and CS3 in Shigella CVD1203, immunized guinea pigs and mice, andshowed that the immunized animals developed high titer of tears secretedIgA (similar to mucosal sIgA), and serum IgG antibodies, towards theShigella LPS and the flimbrial antigens. The ETEC human recombinant LT(K63) gene was expressed in Shigella CVD1204, a guanine dependentstrain, and showed production of sIgA and serum IgG toward the ShigellaLPS as well as towards the LT-A and LT-B subunits of the LT enterotoxin,following immunization of guinea pigs. Koprowski, et al., (Infect.Immun., 68:4884-92 (2000)) co-expressed in CVD1204 the CFA/I and LT(K63) antigens, and demonstrates production of antibodies of the sIgAand serum IgG toward the Shigella LPS, the CFA/I pili antigens and theLT antigens. Altboum et al, (Attenuated Shigella flexneri 2a ΔguaBAstrain CVD 1204 expressing ETEC CS2 and CS3 fimbriae as a live mucosalvaccine against Shigella and enterotoxigenic Escherichia coli infection,In press) immunized guinea pigs with a mixture of CVD1204 strainsexpressing ETEC CS2 and CS3 fimbria. All the immunized animals developedtears sIgA and serum IgG antibodies against Shigella LPS, CS2 and CS3antigens, agglutinating antibodies against Shigella and ETEC CS2 and CS3strains, and were protected against keratoconjunctivitis caused by eyechallenge with the virulent S. flexneri 2a 2457T strain. Those resultsindicate that a combined immunization with live attenuated Shigellastrains expressing ETEC fimbria might induce protection againstShigellosis and ETEC infection.

SUMMARY OF THE INVENTION

[0015] The disclosed invention relates to compositions and methods ofusing csa operon products and the nucleotide and amino acid sequencesencoded thereby. One embodiment relates to an immunogenic compositioncomprising a recombinant product of a csa operon and a carrier. Variousaspects of this embodiment relate to compositions in which therecombinant product of the csa operon is CsaA, CsaB, CsaC, CsaD, CsaE,or a product that is at least 95% homologous to anyone of these csaoperon products. Additionally, the recombinant product of the csa operoncan comprise the csa operon itself.

[0016] Another embodiment relates to an isolated nucleotide sequencecomprising a csa operon or a functional fragment thereof. Anotherembodiments relates to a purified polypeptide sequence expressed from arecombinant csa operon.

[0017] Also disclosed are various methods of using the csa operon,products, and fragments thereof. One embodiment teaches a method ofgenerating an immune response, comprising providing an immunogeniccomposition to a subject, wherein the immunogenic composition comprisesa csa operon, a functional fragment thereof, or product thereof, andcontacting the subject with the immunogenic composition, whereby animmune response is generated in the subject.

[0018] Another embodiment relates to a method of producing a polypeptideproduct from a csa operon or functional fragment thereof, comprisingproviding the csa operon in an expression vector, introducing theexpression vector into a host cell, such that a recombinant host cell isproduced, and subjecting the recombinant host cell to conditions such aprotein from the csa operon is expressed.

[0019] Additional embodiment encompass cells containing recombinant thecsa operon or fragments thereof and vectors comprising the csa operon orfragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. Plasmid constructs containing the entire csa operon.Plasmid pKS-CSA-I contains the csaB, csaC, csaE, csaD′ genes, and ISIelement, (1A). Plasmid pKS-CSA-II contains the csaA genes and upstreamsequences flanked with IS21 element.(1B). Schematic representation ofthe csa operon, (1C).

[0021]FIG. 2. The CS4 fimbriae expression plasmid. Schematic descriptionof the stabilized plasmid pGA2, (FIG. 2A), that is maintained with 15copies per cell, and contained the the hok-sok post-segregationalkilling system, the parA and parM plasmid partitioning system; the aphallele for resistance to kanamycin, and multiple cloning sites. FIG. 2B,Plasmid pGA2-CS4 that contained the cloned CS4 fimbriae encoding genes,csaA, csaB, csaC, and csaE.

[0022]FIG. 3. Alignment of the amino acid sequence of CsaE to otherhomologous proteins. The predicted amino acid sequence of CsaE (SEQ IDNO:28) based on the gene sequence was used in a BLAST search for otherhomologous proteins. CfaE (SEQ ID NO:29) is the tip adhesion moiety ofCS2; CooD (SEQ ID NO:30) is the tip adhesion moiety of CS1; and TsaD(SEQ ID NO:31) is the adhesion protein from Salmonella typhi. CotD (SEQID NO:32) is the spore coat protein of Bacillus subtilis.

[0023]FIG. 4. Alignment of the amino acid sequence of CsaB to other ETECfimbrial subunit proteins. The predicted amino acid sequence of CsaB(SEQ ID NO:33) based on the DNA sequence was used in a BLAST search toidentify homologous proteins. The other fimbrial subunits were CfaA (SEQID NO:34), CooA (SEQ ID NO:35), CotA (SEQ ID NO:36), CsuA1 (SEQ IDNO:37), CsuA2 (SEQ ID NO:38), CsdA (SEQ ID NO:39), and CsbA (SEQ IDNO:40).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The description below relates to the csa operon, which encodesthe proteins required for the production of CS4 antigen, and thegeneration of immunogenic compositions containing products or fragmentsof the csa operon. Methods of making and using the disclosed immunogeniccompositions are also described.

[0025] To generate the disclosed immunogenic compositions, the csaoperon has been sequenced and cloned into an expression vector. Thegenes of the csa operon have been expressed in Shigella CVD1204,resulting in the production of CS4 in this recombinant organism.Preliminary results from immunization of guinea pigs with thisrecombinant strain indicate the development of an immune responseagainst both Shigella and the CS4 pili. Accordingly, the work describedherein relating to the csa operon is applicable to the formulation andadministration of immunogenic compositions with activity against avariety of diseases.

[0026] Organization of the csa Operon

[0027] The csa operon is located on a DNA fragment of approximately ten(10) kilobases in length. The operon comprises 5 genes that are flankedon both sides by insertion elements. This structure is similar to thepathogenicity island described in FIG. 1.

[0028] From the entire fragment, 7245 base pairs were sequenced on bothstrands and the data are presented in FIG. 2. The size of the csa operonwas found to be approximately 6099 base pairs in length and encompassesfive open reading frames.

[0029] Sequence homology to genes encoding the subunits of other ETECfimbriae for which biochemical analysis is available indicates thefollowing functions for each of the open reading frames. The csaA geneencodes the CsaA protein, which is hypothesized to be a periplasmicchaperon-like protein. The csaB gene encodes the CsaB protein, which ishypothesized to be the major pilin subunit. Amino terminal sequenceanalysis of the CS4 pilin subunit confirms the role for this gene. ThecsaC gene encodes the CsaC protein is hypothesized to be a membraneusher protein. The csaD gene encodes a truncated CsaD, which ishypothesized to be a regulatory protein. The csaE gene encodes the CsaEprotein, which is hypothesized to be a tip-associated pilin protein. Theamino acid sequences of the CsaA-E proteins are described in FIG. 3.

[0030] The location of the individual genes in the csa operon, and theproperties of the CsaA-E proteins, are described in Table 1. TABLE 1Properties of the csa operon. properties of the CS4 proteins: LocationNo. of Calculated Theoretical Signal peptide, Gene (bp) PRO AA MW pI No.of AA csaA 283-999 CsaA 238 27305.6 9.29 19 csaB 1028-1531 CsaB 16717343.9 6.56 23 csaC 1589-4192 CsaC 867 97686.93 8.42 22 csaE 4196-5281CsaE 361 40102.4 8.75 23 csaD 5790-6119 CsaD 109 Missing the N-terminal48 amino acids.

[0031] The amino acid sequences of the CsaA-E proteins share homology tosimilar proteins from other ETEC fimbriae. A comparison of proteinhomologies is shown in Table 2. TABLE 2 Homology of CsaA-E proteins toother ETEC fimbriae proteins. Homology to: CS4 NCBI Pro Pili Protein no.Identities Positive Gaps CsaA CFA/I CfaA M55661 208/238 (87%) 218/238(91%) CS1 CooB P25731 124/221 (56%) 161/221 (72%) 4/221 (1%) CS2 CotBS57934 111/238 (46%) 157/238 (65%) 3/238(1%) CsaB CS4 CsfA X97493109/134 (81%) 110/134 (81%) CFA/I CfaB P02971 99/170 (58%) 122/170 (71%)3/170(1%) CS14 CsuA1 X97491 95/164 (57%) 116/164 (69%) CS1 CooA P2573085/168 (50%) 108/168 (63%) 4/168 (2%) CS2 CotA S57935 76/167 (45%)105/167 (62%) 3/167 (1%) CS14 CsuA2 X97492 67/132 (50%) 84/132 (62%)1/132 (1%) CS19 CsdA X97494 56/131 (42%) 75/131 (56%) 1/132 (0%) CS17CsbA X97495 54/132 (40%) 75/132 (55%) 1/132 (0%) CsaC CFA/I CfaC P25733800/868 (92%) 821/868 (94%) CS1 CooC S49538 531/841 (63%) 659/841 (78%)3/841 (0%) CS2 CotC S57936 469/839 (55%) 610/839 (71%) 3/839 (0%) CsaECFA/I CfaE P25734 268/361 (74%) 293/361 (80%) 1/361 (0%) CS1 CooD S49539177/329 (59%) 224/329 (67%) 11/329 3%) CS2 CotD S57937 161/339 (47%)218/339 (63%) 7/339 (2%) CsaD CFA/I CfaD P25393 90/101 (89%) 98/101(96%) RNS P16114 89/101 (88%) 98/101 (96%) CSVR P3460 75/103 (72%)88/103 (84%) AAF/I AGGR P43464 57/101 (56%)

[0032] The csa operon encodes the synthesis of ETEC CS4 fimbriae. Thisoperon was cloned from ETEC E11881A, a CS4 producer strain. A nucleicacid fragment comprising 7239 base pairs was sequenced (in bothdirections). The results indicate that the csa operon is located on anucleic acid fragment 6095 base pairs in length. (Accession No.AF296132).

[0033] The sequence of the csa operon was analyzed. The analysis of theoperon indicated that the csa operon encodes five proteins. Theseproteins are the fimbriae structural protein (CsaB), the tip associatedprotein (CsaE), a chaperon-like protein (CsaA), a usher-like protein(CsaC), and a truncated regulatory protein (CsaD).

[0034] The CsaB protein consist of 167 amino acids, (23 of whichcomprise a signal peptide), producing an ˜17 kDa peptide. The amino acidsequence of the CsaB protein shares homology with other ETEC fimbriaeproteins. For example, CsaB is 71% homologous to the CfaB protein ofCFA/I, 69% and 62% homologous to the two CS 14 structural proteins, 63%homologous to the CS1 structural protein, 62% homologous to the CS2structural protein, 56% homologous to the CS19 structural protein, and55% homologous to the CS17 structural protein.

[0035] The CsaE protein is believed to be a tip associated protein basedon its homology to known tip proteins of other ETEC fimbriae. It is aprotein of 361 amino acids, 23 amino acids of which are cleaved toproduce a globular ˜40 kDa protein. The amino acid sequence of the CsaEprotein shares homology to CFA/I, CS 1 and CS2 pili tip proteins, of80%, 67% and 63%, respectively. The fimbrial assembly proteins CsaA andCsaC share homology to similar proteins from CFA/I, CS1 and CS2 pili.

[0036] Csa is believed to be a regulatory protein based on its homologyto other known ETEC regulatory proteins. Its 48 terminal amino acidswere deleted as a result of an insertion of an IS1 element. In addition,two frame shift mutations following 100 amino acids resulted in a stopcodon. ETEC CFA/I strain E7473 contains a truncated CfaD′ like protein(144 amino acids out of 265), which also contains a stop codon andvarious frame shift mutations. (Jordi, et al., DNA Seq., 2:257-263(1992)). The position of the frame shift mutation in cfaD′ (AccessionAAC41418) is at the same region as in csaD′.

[0037] Upstream to the csaA gene (approximately 3.5 kb) there is anadditional IS21 element, rendering the csa operon flanked between thetwo insertion elements, which is characteristic of a mobile structure.The size of this pathogenicity island-like structure is about 10,500base pairs and its G+C ratio is lower than that of E. coli (38.8% versus50.8%).

[0038] Other ETEC fimbrial operons are carried on similar mobilestructures. The CS1 operon contains IS sequences on both sides (IS150(Accession: X62495), and IS2 (Accession No.: X76908)(Froehlich, et al.,Mol. Microbiol., 12:387-401 (1994)). The CS2 operon contains at itsupstream site IS3 and IS1 DNA sequences, (Accession No.:Z47800)(Froehlich, et al., Infect. Immun., 63:4849-4856 (1995)). The CS6pili contains IS elements, upstream IS91 and IS102, and downstream IS629and IS3, (Accession U04844)(Wolf, et al., FEMS Microbiol. Lett.,148:35-42 (1997)). The CS5 pili operon contains IS1 sequences at theupstream position that are 99% homologous to the IS1 sequences of thecsa operon, and at its downstream site, IS30 sequences. (AccessionAJ224079)(Duthy, et al., J. Bacteriol., 181:5847-5851 (1999)).

[0039] The CS4 major fimbrial protein shares a high degree of amino acidsequence homology to CFA/I, CS1 and CS2 fimbrial proteins.Cross-reaction between antibodies against CS4 and CFA/I, CS1, CS2, andCS17 fimbriae were described by McConnell et al 1989. (McConnell, etal., FEMS Microbiol. Lett., 52:105-108 (1989)).

[0040] The high homology between the structural proteins of CS4 andCFA/I fimbriae resulted in antibodies that cross-reacted with bothfimbriae. Monoclonal antibodies against CFA/I cross reacted with CS4 andinhibited the binding of both fimbriae containing strains to humanjejunal enterocytes and to Caco-2 cells. These antibodies also inhibitedhemagglutination and conferred passive protections against fluidaccumulation in rabbit ileal loops caused by infection of both ETECstrains. (Rudin, et al., Microb. Pathog., 21:35-45 (1996)). Moreover, ithas been hypothesized that immunization with purified CFA/I and CS4fimbriae may prime and boost immune response against the homologous andheterologous fimbriae. (Rudin and Svennerholm, Microb. Pathog.,16:131-139 (1994); Rudin, et al., Epidemiol. Infect., 119:391-393(1997)).

[0041] Nucleotide Sequences Relating to the csa Operon

[0042] Having identified the csa operon and the genes encoded thereby,this knowledge has been used to produce useful immunogenic compositions.As is discussed more fully in the Examples below, the genes of the csaoperon were cloned, sequenced and expressed. Polynucleotide moleculesencoding the proteins of the csa operon and their sequences are providedbelow.

[0043] Representative polynucleotide molecules encoding the proteins ofthe csa operon (SEQ ID. NO.: 27) include sequences comprising csaA (SEQ.ID. NO.: 1), csaB (SEQ. ID. NO.: 3), csaC (SEQ. ID. NO.: 5), csaD (SEQ.ID. NO.: 7), and csaE (SEQ. ID. NO.: 9). Polynucleotide moleculesencoding the proteins of the csa operon include those sequencesresulting in minor genetic polymorphisms, differences between strains,and those that contain amino acid substitutions, additions, and/ordeletions.

[0044] In some instances, one can employ such changes in the sequence ofa recombinant csa operon-encoded protein to substantially decrease orincrease the biological activity of a particular csa operon-encodedprotein relative to the activity of the corresponding wild-type csaoperon-encoded protein. Such changes can also be directed towards anendogenous csa operon encoded sequence using, for example, variousmolecular biological techniques to alter the endogenous gene andtherefore its protein product.

[0045] Nucleotide sequences encoding csa operon proteins can be used toidentify polynucleotide molecules encoding other proteins withbiological functions similar to that of the csa operon. ComplementaryDNA molecules encoding csa operon-like proteins can be obtained byconstructing a cDNA library from mRNA from eukaryotic cells or a DNAlibrary from other prokaryotic organisms. DNA molecules encoding csaoperon-like proteins can be isolated from such a library using thesequences disclosed herein with standard hybridization techniques or bythe amplification of sequences using polymerase chain reaction (PCR)amplification.

[0046] In a similar manner, genomic DNA encoding csa operon proteinhomologs can be obtained using probes designed from the sequencesdisclosed herein. Suitable probes for use in identifying csa operonproduced protein homologue sequences can be obtained from csaoperon-specific sequences. Alternatively, oligonucleotides containingspecific DNA sequences from a csa operon-coding region can be used toidentify related csa clones. One of ordinary skill in the art willappreciate that the regulatory regions of the csa operon and homologousgenes and operons can be obtained using similar methods.

[0047] csa operon homologous polynucleotide molecules can be isolatedusing standard hybridization techniques with probes of at least about 7nucleotides in length and up to and including the full coding sequence.Homologous csa operon sequences can be identified using degenerateoligonucleotides capable of hybridization based on the sequencesdisclosed herein for use PCR amplification or by hybridization atmoderate or greater stringency. The term, “capable of hybridization” asused herein means that the subject nucleic acid molecules (whether DNAor RNA) anneal to an oligonucleotide of 15 or more contiguousnucleotides of SEQ. ID. NOs: 1, 3, 5, 7, and 9.

[0048] The choice of hybridization conditions will be evident to oneskilled in the art and will generally be guided by the purpose of thehybridization, the type of hybridization (DNA-DNA or DNA-RNA), and thelevel of desired relatedness between the sequences. Methods forhybridization are well established in the literature. One of ordinaryskill in the art realizes that the stability of nucleic acid duplexeswill decrease with an increased number and location of mismatched bases;thus, the stringency of hybridization can be used to maximize orminimize the stability of such duplexes. Hybridization stringency can bealtered by: adjusting the temperature of hybridization; adjusting thepercentage of helix-destabilizing agents, such as formamide, in thehybridization mix; and adjusting the temperature and salt concentrationof the wash solutions. In general, the stringency of hybridization isadjusted during the post-hybridization washes by varying the saltconcentration and/or the temperature, resulting in progressively higherstringency conditions.

[0049] An example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).Washing can be carried out using only one of these conditions, e.g.,high stringency conditions, or each of the conditions can be used, e.g.,for 10-15 minutes each, in the order listed above, repeating any or allof the steps listed. As mentioned above, however, optimal conditionswill vary, depending on the particular hybridization reaction involved,and can be determined empirically. In general, conditions of highstringency are used for the hybridization of the probe of interest.

[0050] Alternatively, polynucleotides having substantially the samenucleotide sequence set forth in SEQ. ID. NOs: 1, 3, 5, 7, and 9 orfunctional fragments thereof, or nucleotide sequences that aresubstantially identical to SEQ. ID. NOs: 1, 3, 5, 7, and 9, canrepresent members of a csa-like operon. By “substantially the same” or“substantially identical” is meant a nucleic acid or polypeptideexhibiting at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% homology to a reference nucleic acid. For nucleotidesequences, the length of comparison sequences will generally be at least10 to 500 nucleotides in length. More specifically, the length ofcomparison will be at least 50 nucleotides, at least 60 nucleotides, atleast 75 nucleotides, and at least 110 nucleotides in length.

[0051] One embodiment of the invention provides isolated and purifiedpolynucleotide molecules encoding one or more csa operon proteins,wherein the polynucleotide molecules that are capable of hybridizingunder moderate to stringent conditions to an oligonucleotide of 15 ormore contiguous nucleotides of SEQ. ID. NOs: 1, 3, 5, 7, and 9,including complementary strands thereto.

[0052] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridization orcomputer-based techniques which are well known in the art. Suchtechniques include, but are not limited to: 1) hybridization of genomicor cDNA libraries with probes to detect homologous nucleotide sequences;2) antibody screening of expression libraries to detect cloned DNAfragments with shared structural features; 3) polymerase chain reaction(PCR) on genomic DNA or cDNA using primers capable of annealing to theDNA sequence of interest; 4) computer searches of sequence databases forsimilar sequences; and 5) differential screening of a subtracted DNAlibrary.

[0053] Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of csa operon sequences provided herein andencoding a Csa protein, can be synthesized chemically. This synthesisrequires that short, oligo-peptide stretches of the amino acid sequencebe known. The DNA sequence encoding the protein can be deduced from thegenetic code, however, the degeneracy of the code must be taken intoaccount. It is possible to perform a mixed addition reaction when thesequence is degenerate. This includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of MRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA or DNA cloneby the hybridization of the target DNA to that single probe in themixture that is its complete complement. (Wallace, et al., Nucl. AcidRes., 9:879, 1981). Alternatively, a subtractive library is useful forelimination of non-specific cDNA clones.

[0054] Among the standard procedures for isolating DNA sequences ofinterest is the formation of plasmid- or phage-carrying genomiclibraries which include total DNA from the organism of interest. Whenused in combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target DNAcan be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the DNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

[0055] The nucleotide sequences of the disclosed herein have a myriad ofapplications. Representative uses of the nucleotide sequences of theinvention include the construction of DNA and oligonucleotide probesuseful in Northern, Southern, and dot-blot assays for identifying andquantifying the level of expression of csa operon encoded proteins in acell. csa operon encoded proteins have a variety of uses, for example,as antigens with which to elicit an immune response.

[0056] In addition, considering the important role the CS4 pili plays inETEC attachment and colonization, it is thought highly likely thatcompositions containing the CS4 pili or compositions with activityagainst the same can result from expression of the csa operon. In thiscase, the proteins of the csa operon can prove highly useful in thegeneration of immunogenic compositions that can be used to generate animmune response in a subject.

[0057] Similarly, csa operon nucleotide sequences can be employed forthe construction of recombinant cell lines, recombinant organisms,expression vectors, and the like. Such recombinant constructs can beused to express recombinant csa operon proteins. In one embodiment, therecombinant constructs can be used to screen for candidate therapeuticagents capable of altering the pathology of a CS4 antigen-expressingorganism. In another embodiment, the proteins of the csa operon presentan attractive set of proteins with which to create immunogeniccompositions. For example, the CS4 pili can be expressed, purified, andused to prepare an immunogenic subunit composition. In anotherembodiment, recombinant CS4 pili can be expressed in an organism, andthe whole organism can be formulated into an immunogenic composition.

[0058] When the coding regions of the csa operon are used in theconstruction of various types of vectors, the csa sequences are ofteninserted into the coding region of the vector under the control of apromoter. Additionally, other elements, including regulatory elements,which are commonly found in vectors suitable for use in variousmolecular biology techniques, can also be included.

[0059] In one embodiment, a vector comprising a DNA molecule encoding aCsa protein is provided. Preferably, a DNA molecule including a csaA,csaB, csaC, csaD, or csaE gene, or a combination of these genes isinserted into a suitable expression vector, which is in turn used totransfect or transform a suitable host cell. Exemplary expressionvectors include a promoter capable of directing the transcription of apolynucleotide molecule of interest in a host cell. Representativeexpression vectors include both plasmid and/or viral vector sequences.Suitable vectors include retroviral vectors, vaccinia viral vectors, CMVviral vectors, BLUESCRIPT (Stratagene, San Diego, Calif.) vectors,bacculovirus vectors, and the like. In another embodiment, promoterscapable of directing the transcription of a cloned gene or cDNA can beinducible or constitutive promoters and include viral and cellularpromoters.

[0060] In some embodiments, it can be preferable to use a selectablemarker to identify cells that contain the cloned DNA. Selectable markersare generally introduced into the cells along with the cloned DNAmolecules and include genes that confer resistance to drugs, such asampicillin, neomycin, hygromycin, and methotrexate. Selectable markerscan also complement auxotrophies in the host cell. Other selectablemarkers provide detectable signals, such as beta-galactosidase toidentify cells containing the cloned DNA molecules.

[0061] Antisense

[0062] Antisense csa nucleotide sequences can be used to block csaexpression. Suitable antisense oligonucleotides are at least 11nucleotides in length and can include untranslated (upstream) andassociated coding sequences. As will be evident to one skilled in theart, the optimal length of an antisense oligonucleotide depends on thestrength of the interaction between the antisense oligonucleotide andthe complementary mRNA, the temperature and ionic environment in whichtranslation takes place, the base sequence of the antisenseoligonucleotide, and the presence of secondary and tertiary structure inthe mRNA and/or in the antisense oligonucleotide. Suitable targetsequences for antisense oligonucleotides include promoter regions,ribosome binding sites, and sites that interfere with ribosomeprogression.

[0063] Antisense oligonucleotides can be prepared, for example, by theinsertion of a DNA molecule containing the target DNA sequence into asuitable expression vector such that the DNA molecule is inserteddownstream of a promoter in a reverse orientation as compared to theparticular csa gene itself. The expression vector can then betransduced, transformed or transfected into a suitable cell resulting inthe expression of antisense oligonucleotides. Alternatively, antisenseoligonucleotides can be synthesized using standard manual or automatedsynthesis techniques. Synthesized oligonucleotides are introduced intosuitable cells by a variety of means including electroporation, calciumphosphate precipitation, or microinjection. The selection of a suitableantisense oligonucleotide administration method will be evident to oneskilled in the art.

[0064] With respect to synthesized oligonucleotides, the stability ofantisense oligonucleotide-mRNA hybrids is advantageously increased bythe addition of stabilizing agents to the oligonucleotide. Stabilizingagents include intercalating agents that are covalently attached toeither or both ends of the oligonucleotide. In preferred embodiments,the oligonucleotides are made resistant to nucleases by, for example,modifications to the phosphodiester backbone by the introduction ofphosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates,phosphoramidates, phosphorodithioates, or morpholino rings.

[0065] Amino Acids

[0066] The identification of the csa operon and the proteins encodedthereby is described. Representative polypeptides produced from thegenes of the csa operon include sequences comprising CsaA (SEQ. ID. NO.:2), CsaB (SEQ. ID. NO.: 4), CsaC (SEQ. ID. NO.: 6), CsaD (SEQ. ID. NO.:8), and CsaE (SEQ. ID. NO.: 10). Variants of the csa operon encodedproteins include those amino acid sequences resulting from in minorgenetic polymorphisms, differences between strains, and those thatcontain amino acid substitutions, additions, and/or deletions.

[0067] The csa operon encoded proteins, as described herein, encompassthe whole proteins encoded by the operon, as well as fragments of csaproteins that are functionally active. csa operon encoded proteinspurified from naturally occurring materials and closely related,functionally similar proteins retrieved by antisera specific to the csaproteins, and recombinantly expressed proteins encoded by geneticmaterials (DNA, RNA, cDNA) retrieved on the basis of their similarity toregions in the csa operon sequences are also encompassed by the presentdescription.

[0068] According to the present description, polynucleotide moleculesencoding csa operon encoded proteins encompass those molecules thatencode csa proteins or peptides that share identity with the sequencesshown in SEQ. ID. NOs.: 2, 4, 6, 8, and 10. Such molecules preferablyshare greater than 30% identity at the amino acid level with thedisclosed sequences in csa. In preferred embodiments, the polynucleotidemolecules can share greater identity at the amino acid level acrosshighly conserved regions.

[0069] It is contemplated that amino acid sequences substantially thesame as the sequences set forth in SEQ. ID. NOs.: 2, 4, 6, 8, and 10,are encompassed by the present description. A preferred embodimentincludes polypeptides having substantially the same sequence of aminoacids as the amino acid sequence set forth in SEQ ID NOs.: 2, 4, 6, 8,and 10, or functional fragments thereof, or amino acid sequences thatare substantially identical to SEQ ID NOs.: 2, 4, 6, 8, and 10. By“substantially the same” or “substantially identical” is meant apolypeptide exhibiting at least 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% homology to a reference amino acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids.

[0070] Homology is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705 or the NCBI BLAST program). Such softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, substitutions, and other modifications.

[0071] The term “functional fragments” include those fragments of SEQ IDNOs.: 2, 4, 6, 8, and 10, or other proteins that have a similar aminoacid sequence as that of the csa operon encoded proteins, that retainthe function or activity of the various csa proteins. One of skill inthe art can screen for the functionality of a fragment by using theexamples provided herein, where full-length csa operon encoded proteinsare described. It is also envisioned that fragments of various csaoperon encoded proteins can be identified in a similar manner.

[0072] By “substantially identical” is also meant an amino acid sequencewhich differs only by conservative amino acid substitutions, forexample, substitution of one amino acid for another of the same class(e.g., valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein assayed, (e.g., as described herein). Preferably, such asequence is at least 85%, and more preferably from 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, to 100% homologous at the amino acid levelto SEQ ID NOs:2, 4, 6, 8, or 10.

[0073] By a “substantially pure polypeptide” is meant a csa operonencoded protein that has been separated from components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, by weight, free from the proteins and other naturallyoccurring molecules with which it is typically associated. Preferably,the preparation is at least 75%, 80%, 90%, 95%, and most preferably atleast 99%, by weight, csa operon encoded protein. A substantially purecsa operon encoded polypeptide can be obtained, for example, byextraction from a natural source; by expression of a recombinant nucleicacid encoding a csa operon encoded polypeptide; or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, e.g., column chromatography, polyacrylamide gel electrophoresis,or by HPLC analysis.

[0074] A protein is substantially free of naturally associatedcomponents when it is separated from those contaminants that accompanyit in its natural state. Thus, a protein that is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptidesinclude those derived from eukaryotic organisms but synthesized in E.coli or other prokaryotes.

[0075] As would be evident to one skilled in the art, the polynucleotidemolecules of the present disclosure can be expressed in a variety ofprokaryotic and eucaryotic cells using regulatory sequences, vectors,and methods well established in the literature.

[0076] csa operon encoded proteins produced according to the presentdescription can be purified using a number of established methods suchas affinity chromatography using an anti-Csa protein antibodies coupledto a solid support. Fusion proteins of an antigenic tag and a csa operonencoded protein can be purified using antibodies to the tag. Optionally,additional purification is achieved using conventional purificationmeans such as liquid chromatography, gradient centrifugation, and gelelectrophoresis, among others. Methods of protein purification are knownin the art and can be applied to the purification of recombinant csaoperon encoded proteins described herein. Purification of csa operonproducts is discussed more completely below.

[0077] Construction of csa operon encoded fusion proteins is alsocontemplated. Fusion proteins will typically contain additions,substitutions, or replacements of one or more contiguous amino acids ofthe native csa operon encoded protein with amino acid(s) from a suitablefusion protein partner. Such fusion proteins are obtained usingrecombinant DNA techniques well known by one of skill in the art.Briefly, DNA molecules encoding the hybrid csa operon encoded proteinsof interest are prepared using generally available methods such as PCRmutagenesis, site-directed mutagenesis, and/or restriction digestion andligation. The hybrid DNA is then inserted into expression vectors andintroduced into suitable host cells.

[0078] One embodiment of the present invention involves the isolation ofproteins that interact with csa operon encoded proteins or are receptorsfor CS4 pili. csa operon encoded proteins can be used inimmunoprecipitation to isolate interacting factors or used for thescreening of interactors using different methods of two hybridscreening. Isolated interactors of csa operon encoded proteins can beused to modify or block CS4 mediated binding to a host cell.

[0079] Synthetic peptides, recombinantly derived peptides, fusionproteins, chiral proteins (stereochemical isomers, racemates,enantiomers, and D-isomers) and the like are provided which include aportion of a csa operon encoded protein or the entire protein. Thesubject peptides have an amino acid sequence encoded by a nucleic acidwhich hybridizes under stringent conditions with an oligonucleotide of15 or more contiguous nucleotides of SEQ. ID. NOs: 1, 3, 5, 7, and 9.Representative amino acid sequences of the subject peptides aredisclosed in SEQ. ID. NOs: 2, 4, 6, 8, and 10. The subject peptides finda variety of uses, including preparation of specific antibodies andpreparation of antagonists of CS4 binding.

[0080] Antibodies

[0081] As noted above, the described teachings provide antibodies thatbind to csa operon encoded proteins. The production of non-humanantisera or monoclonal antibodies (e.g., murine, lagomorph, porcine,equine) is well known and can be accomplished by, for example,immunizing an animal with a csa operon encoded protein or peptides. Forthe production of monoclonal antibodies, antibody producing cells areobtained from immunized animals, immortalized and screened, or screenedfirst for the production of the antibody that binds to the particularcsa operon encoded protein or peptides and then immortalized. It can bedesirable to transfer the antigen binding regions (e.g., F(ab′)2 orhypervariable regions) of non-human antibodies into the framework of ahuman antibody by recombinant DNA techniques to produce a substantiallyhuman molecule.

[0082] Following synthesis or expression and isolation or purificationof a csa operon encoded protein or a portion thereof, the isolated orpurified protein can be used to generate antibodies and tools foridentifying agents that interact with the csa operon encoded protein andfragments of interest. Depending on the context, the term “antibodies”can encompass polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by a Fab expression library. Antibodiesthat recognize csa operon encoded proteins and fragments thereof havemany uses including, but not limited to, biotechnological applications,therapeutic/prophylactic applications, and diagnostic applications.

[0083] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc., can be immunized by injection with csa operonencoded proteins or any portion, fragment or oligopeptide that retainsimmunogenic properties. Depending on the host species, various adjuvantscan be used to increase immunological response. Such adjuvants include,but are not limited to, detoxified heat labile toxin from E. coli,Freund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacillus Calmette-Guerin) and Corynebacterium parvum are alsopotentially useful adjuvants.

[0084] Peptides used to induce specific antibodies can have an aminoacid sequence consisting of at least three amino acids, and preferablyat least 10 to 15 amino acids. Preferably, short stretches of aminoacids encoding fragments of csa operon encoded proteins are fused withthose of another protein such as keyhole limpet hemocyanin such that anantibody is produced against the chimeric molecule. While antibodiescapable of specifically recognizing csa operon encoded proteins can begenerated by injecting synthetic 3-mer, 10-mer, and 15-mer peptides thatcorrespond to a protein sequence of the csa operon encoded protein orproteins of interest into mice, a more diverse set of antibodies can begenerated by using recombinant csa operon encoded proteins, purified csaoperon encoded proteins, or fragments of csa operon encoded proteins.

[0085] To generate antibodies to csa operon encoded proteins andfragments thereof, a substantially pure csa operon encoded protein or afragment thereof is isolated from a transfected or transformed cell orthe wildtype ETEC organism. The concentration of the polypeptide in thefinal preparation is adjusted, for example, by concentration on anAmicon filter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibody to the polypeptide of interest can then be preparedas follows:

[0086] Monoclonal antibodies to csa operon encoded proteins or afragment thereof can be prepared using any technique that provides forthe production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (Nature 256:495-497 (1975),the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72(1983); Cote et al Proc Natl. Acad. Sci 80:2026-2030 (1983), and theEBV-hybridoma technique Cole et al. Monoclonal Antibodies and CancerTherapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985). In addition,techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activitycan be used. (Morrison et al. Proc Natl. Acad. Sci 81:6851-6855 (1984);Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature314:452-454(1985). Alternatively, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce csa operon encoded protein-specific single chainantibodies. Antibodies can also be produced by inducing in vivoproduction in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in Orlandi et al., Proc Natl. Acad. Sci 86: 3833-3837(1989), and Winter G. and Milstein C; Nature 349:293-299 (1991).

[0087] Antibody fragments that contain specific binding sites for csaoperon encoded proteins can also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments thatcan be produced by pepsin digestion of the antibody molecule and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (Huse W. D. et al. Science256:1275-1281 (1989)).

[0088] By one approach, monoclonal antibodies to csa operon encodedproteins or fragments thereof are made as follows. Briefly, a mouse isrepetitively inoculated with a few micrograms of the selected protein orpeptides derived therefrom, over a period of a few weeks. The mouse isthen sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused in the presence of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, E., Meth. Enzymol. 70:419 (1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Davis, L. et al. Basic Methods inMolecular Biology Elsevier, N.Y. Section 21-2.

[0089] Polyclonal antiserum containing antibodies to heterogeneousepitopes of a single protein can be prepared by immunizing suitableanimals with the expressed protein or peptides derived therefromdescribed above, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Forexample, small molecules tend to be less immunogenic than others and canrequire the use of carriers and adjuvant. Also, host animals vary inresponse to site of inoculations and dose, with both inadequate orexcessive doses of antigen resulting in low titer antisera. Small doses(ng level) of antigen administered at multiple intradermal sites appearsto be most reliable. An effective immunization protocol for rabbits canbe found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab.33:988-991 (1971).

[0090] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively (e.g., in diagnostic embodimentsthat identify the presence of a csa operon encoded protein in biologicalsamples). It is also contemplated that various methods of molecularmodeling and rational drug design can be applied to identify compoundsthat resemble a csa operon encoded protein, fragment, or derivativethereof, and molecules that interact with csa operon encoded proteinsand, thereby modulate their function.

[0091] Expression Vectors

[0092] Recombinant gene expression vectors comprising the csa operon, orportions thereof, can be constructed in a variety of forms well-known inthe art. Preferred expression vectors include plasmids and cosmids.Expression vectors include one or more fragments of the csa operon.Typically, an expression vector will comprise one or more genes of csaoperon. In one embodiment, an expression vector will comprise andoperatively encode the csaA, csaB, csa C, csaE, and csaD coding regions.Alternative embodiments of the described expression vectors can havevarious combinations of the coding regions csa operon. For example, anexpression can comprise the csaB, the csaE coding regions, or acombination of both.

[0093] As used herein, the phrase “operatively encode” refers to one ormore protein coding regions associated with those regulatory sequencesrequired for expression of the polypeptide encoded by the coding region.Examples of such regulatory regions including promoter binding sites,enhancer elements, ribosome binding sites, and the like. Those ofordinary skill in the art will be able to select regulatory sequencesand incorporate them into the recombinant expression vectors describedherein without undue experimentation. For example, suitable regulatorysequences for use in various eukaryotic and prokaryotic systems aredescribed in Ausubel, et al., Short Protocols in Molecular Biology,3^(rd) ed., John Wiley & Sons, Inc, New York, 1997, which is herebyincorporated by reference in its entirety.

[0094] Expression vectors for use with the csa operon will typicallycontain regulatory sequences derived from a compatible species forexpression in the desired host cell. For example, when E. coli is thehost cell, the host cell population can be typically transformed usingpBR322, a plasmid derived from an E. coli species. (Bolivar, et al.,Gene, 2:95, 1977). pBR322 contains genes for ampicillin (AMPR) andtetracycline resistance and thus provides easy means for identifyingtransformed cells.

[0095] Promoters suitable for use with prokaryotic hosts illustrativelyinclude the beta-lactamase and lactose promoter systems (Chang, et al.,Nature, 275:615, 1978; and Goeddel, et al., Nature, 281:544, 1979),alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8:4057, 1980) and hybrid promoters such as the taqpromoter (de Boer, et al., Proc. Natl. Acad. Sci. USA, 80:21-25, 1983).Other functional bacterial promoters are also suitable. Their nucleotidesequences are generally known in the art, thereby enabling a skilledworker to ligate them to a polynucleotide which encodes the peptide ofinterest (Siebenlist, et al., Cell, 20:269, 1980) using linkers oradapters to supply any required restriction sites.

[0096] In addition to prokaryotes, eukaryotic microbes such as yeastcultures can also be used as source for the regulatory sequences.Saccharomyces cerevisiae is a commonly used eukaryotic hostmicroorganism. Suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman, et al.,J. Biol. Chem., 255:2073, 1980) or other glycolytic enzymes (Hess, etal. J. Adv. Enzyme Reg. 7:149, 1968; and Holland, Biochemistry, 17:4900,1978) such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0097] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degraded enzymes associated with nitrogen metabolism,metallothionine, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Yeast enhancers alsoare advantageously used with yeast promoters.

[0098] In another embodiment, a recombinant virus is used as theexpression vector. Exemplary viruses include the adenoviruses,adeno-associated viruses, herpes viruses, vaccinia, or an RNA virus suchas a retrovirus or an alphavirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Preferably the alphavirusvector is derived from Sindbis or Semliki Forest Virus. All of thesevectors can transfer or incorporate a gene for a selectable marker sothat transduced cells can be identified and generated.

[0099] By inserting one or more sequences of interest into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, for example, the vector is now targetspecific. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector, such as to the vicinity of a mucosalinductor site, using a MALT-specific antibody. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the polynucleotides of interest.

[0100] It will be appreciated that the same techniques that are utilizedto incorporate the nucleotide sequences of the csa operon and optionallyother immunostimulatory polynucleotides into viral gene expressionvectors can be used to incorporate the sequences into live andattenuated live viruses for use as immunogenic compositions.

[0101] Targeting of mucosal tissues can be performed by exploitinginherent biological properties of the lymphoid bed which is to betargeted. These include the crypt architecture of the tonsillar pillarswhich can be used to entrap particles, and also include the M cells ofPeyer's patches in the gut, which M cells specifically endocytose a widevariety of particles including lipid particles and other smallparticulates. Therefore, those skilled in the art can prepare a widevariety of molecular particulate preparations which, if provided tointestine, will lodge within the crypt portions of intestinal Peyer'spatches and be endocytosed by M cells. If such particles provide fordelivery of a biologically active polynucleotide to M cells, then suchparticles will enable the stimulation or modulation of mucosal immuneresponse induction by the Peyer's patch lymphoid tissue to which the Mcell traffics.

[0102] Construction of suitable vectors containing desired coding,non-coding and control sequences employ standard ligation techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to construct the plasmids required.

[0103] For example, for analysis to confirm correct sequences inplasmids constructed, the ligation mixtures can be used to transform ahost cell and successful transformants selected by antibiotic resistancewhere appropriate. Plasmids from the transformants are prepared,analyzed by restriction and/or sequenced by, for example, the method ofMessing, et al., (Nucleic Acids Res., 9:309, 1981), the method of Maxam,et al., (Methods in Enzymology, 65:499, 1980), or other suitable methodswhich will be known to those skilled in the art. Size separation ofcleaved fragments is performed using conventional gel electrophoresis asdescribed, for example, by Maniatis, et al., (Molecular Cloning, pp.133-134, 1982).

[0104] Host cells can be transformed with the expression vectorsdescribed herein and cultured in conventional nutrient media modified asis appropriate for inducing promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

[0105] Purification

[0106] The purification of one or more of the products of the csa operonis disclosed below. Steps involved in the purification of csa operonproducts include (1) solubilization of the desired protein, (2) thedevelopment of one or more isolation and concentration procedures, (3)stabilization of the protein following purification, and (4) developmentof a suitable assay to determine the presence of the desired protein.Various aspects of protein isolation and purification are discussed indetail in Cooper, T. G., “The Tools of Biochemistry,” John Wiley & Sons,New York, 1977, which is hereby incorporated by reference in itsentirety. As the techniques of protein isolation and purification arenotoriously well known in the art, this disclosure will refrain fromdiscussing them in detail. Nevertheless, elements of the cited referenceare summarized and discussed below.

[0107] Solubilization is required of most proteins to be purified, asmost isolation procedures commonly used operate in aqueous solutions. Insome cases solubilization can be achieved by merely lysing a host cellwithin which a desired protein has been expressed. In other situations,additional steps, such as extracting the desired protein from asubcellular organelle may be required. Osmotic lysis, grinding, the useof blenders, ultrasonic waves, presses, and other well known techniquesof protein solubilization are contemplated for use with the methodsdisclosed herein.

[0108] Regarding the isolation and concentration of csa operon products,there are variety of techniques available that are well known in theart. These techniques include (1) differential solubility, (2) ionexchange chromatography, (3) absorption chromatography, (4) molecularsieve techniques, (5) affinity chromatography, (6) electrophoresis, and(7) electrofocusing. Each of these techniques can be useful in thepurification of one or more csa operon products. An immunogenicpurification methodology for the purification of the CS4 pili isdescribed in Wolf, et al., “Characterization of CS4 and CS6 antigeniccomponents of PCF8775, a putative colonization factor complex fromenterotoxigenic Escherichia coli E8775,” Infect Immun. 57(1):164-73(1989), which is hereby incorporated by reference in its entirety. Forpurifying intact CS4 fimbriae, use the methods described in Hall, etal., J. Bacteriol. 171:6372 (1989).

[0109] Stabilizing and maintaining a purified product of the csa operonin a functional state warrants attention to a number of differentconditions. These conditions include (1) pH, (2) degree of oxidation,(3) heavy metal concentration, (4) medium polarity, (5) proteaseconcentration, and (6) temperature. One of ordinary skill in the artwould readily know which of the available techniques to use to maintainpurified csa operon products in an active form without undueexperimentation.

[0110] Developing one or more assays with which to determine thepresence and functionality of the purified product of the csa operonwill hinge on the individual proteins themselves. Perhaps the mostuseful assay to develop involves the generation of one or moreantibodies with which to identify the various products of the csaoperon. The generation of antibodies is discussed herein. Because thenucleotide and amino acid sequences of each of the csa operon encodedgene sequences is known, it would be trivial for one of ordinary skillin the art to generate suitable antibodies for the detect of Csaproteins, using techniques that are notoriously well known.

[0111] Compositions

[0112] The proteins encoded by the csa operon can be used to formulateimmunogenic compositions that facilitate an immune response. Examples ofa typical immune response include a mucosal immune response and asystemic immune response. A variety of embodiments utilizing theproteins of the csa operon are envisioned.

[0113] Recombinant Organisms

[0114] In one embodiment, a nucleotide sequence comprising the csaoperon or a functional fragment thereof is introduced into an exogenousorganism using standard molecular biology techniques well known to thoseof ordinary skill in the art. Exemplary techniques are discussed inAusubel, et al., “Short Protocols in Molecular Biology.” The resultingrecombinant organism can then be used as an immunogen against which animmune response may be engendered. In a preferred embodiment, anattenuated pathogenic organism serves as the exogenous organism. It iscontemplated that an entire recombinant organism or a functionalfragment thereof, such as an isolated membrane fraction, liposome, orthe like, can be used to generate an immunogenic composition.

[0115] For example, one application of the discoveries described hereinis directed to the development of a multivalent hybrid vaccine toprevent both Shigella dysentery and ETEC diarrhea, (Altboum, Z., et al.,Attenuated Shigella flexneri 2a ΔguaBA strain CVD 1204 expressing ETECCS2 and CS3 fimbriae as a live mucosal vaccine against Shigella andenterotoxigenic Escherichia coli infection, In press; Koprowski, et al.,Infect. Immun., 68:4884-92 (2000); Kotloff, et al., Vaccine, 13:495-502(1995); Levine, et al., “Fimbrial vaccines,” In P. Klemm (ed.),Fimbriae: adhesion, biogenics, genetics and vaccines, Boca Raton: CRCPress, 1994; Noriega, et al., Infect. Immun., 64:23-27 (1996)). U.S.Pat. No. 6,190,669, to Noriega, et al., entitled “Attenuated mutants ofsalmonella which constitutively express the Vi antigen,” which is herebyincorporated by reference in its entirety, contains additional teachingrelating to the generation of such chimeric organisms suitable for usein the preparation of an immunogenic composition containing the CS4pili.

[0116] In a preferred embodiment, the ETEC-CS4 fimbrial encoding genesof the csa operon are isolated and used to construct an efficientmultivalent Shigella-ETEC immunogenic composition that will protect fromdiarrhea caused by either Shigella and CS4 expressing ETEC strains. Oneaspect of this embodiment is directed to creating an immunogeniccomposition capable of generating an immune response against LTh, the LTvariant found in human ETEC strains. The immunogenic properties ofcloned CFA/I, CS2, CS3 and LThK63 encoding genes in S. flexneri 2astrain CVD 1204 have been reported. This work has been expanded byincluding the expression of the CS4 pili as an intact fimbriae in bothE. coli and Shigella strains.

[0117] Other multivalent immunogenic compositions effective againstenteric bacteria are contemplated. For example, a multivalentimmunogenic composition against Salmonella spp.; Clostridium spp., suchas Clostridium botulinum; Staphylococcus spp, such as S. aureus;Campylobacter spp., such as C. jejuni; Yersinia spp., such as Y.enterocolitica and Y. pseudotuberculosis; Listeria spp., such as L.monocytogenes; various Vibrio spp., including V. cholerae O1, V.cholerae non-O1, V. parahaemolyticus, V. vulnificus; Clostridium spp.,such as C. perfringens; Bacillus spp., such as B. cereus; Aeromonasspp., such as A. hydrophila; Plesiomonas spp., such as P. shigelloides;Shigella spp.; Streptococcus spp.; various miscellaneous enterics suchas Klebsiella spp.; Enterobacter spp.; Proteus spp.; Citrobacter spp.;Aerobacter spp.; Providencia spp.; Serratia spp.; and members of theenterovirulent Escherichia coli Group (EEC Group) which comprises,enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichiacoli (EPEC), enterohemorrhagic Escherichia coli (EHEC) such asEscherichia coli O157:H7, and enteroinvasive Escherichia coli (EIEC).

[0118] Subunit Vaccines

[0119] Another embodiment described herein relates to the generation ofimmunogenic compositions comprising distinct immunogenic proteins orfragments thereof, or functional fragments of organisms of interest.Such immunogenic compositions are referred to here as subunitimmunogenic composition because at least one of the components of thecomposition is a subunit of an organism, rather than an entire organism.Typically, a subunit immunogenic composition as described hereincomprises one or more immunogenic components.

[0120] In a preferred embodiment, the subunit immunogenic compositiondescribed herein comprise a carrier component and an immunogeniccomponent. Typically, the carrier component will function as a bindingmoiety with which the originating organism uses to bind to and gainentrance into the host organism. One example of such a carrier componentis the CS4 antigen, which is encoded by the csa operon. The CS4 antigenallows ETEC strains expressing this protein complex to bind to themucosa of a human host. In a preferred embodiment the CsaB and/or theCsaE proteins can function as carrier components.

[0121] Although not wishing to be bound by theory, it is hypothesizedthat the subunit immunogenic composition described herein function byexposing the immunogenic component of the subunit immunogeniccomposition to the mucosa, and various immune system components presentthere. In one theory, the generation of a desired immune response by thesubunit immunogenic compositions described herein occurs by increasingthe exposure of the immunogenic compositions to the target tissue. Thepresence of both a carrier component and an immunogenic component aretheorized to achieve this goal.

[0122] Any protein, peptide, or amino acid sequence that elicits animmune response can be used as the immunogenic component in the subunitimmunogenic compositions described herein.

[0123] The carrier components of the described subunit immunogeniccompositions can also possess immunogenic characteristics themselves.Typically, adjuvants are used in immunogenic compositions to enhance theimmune response directed against the immunogenic component of theimmunogenic compositions disclosed. Carrier components that possess bothmucosa binding characteristics and immunogenic characteristics can beused. For example, in one embodiment, the CsaB and/or the CsaE proteinscan function both as the carrier component and the immunogeniccomponent.

[0124] For the carrier components described above, the entire moleculecan be used as the carrier component, or a functionally active fragmentof the molecule can be used. Mutagenized forms of these molecules canalso be used as carrier components.

[0125] In one embodiment, one or more Csa proteins are isolated andpurified. The one or more Csa proteins can be mixed or coupled with oneor more immunogenic compounds. For example, in one embodiment, CsaB canbe expressed, purified, and cross-linked to a toxin, toxoid (anattenuated toxin), or some other immunogenic compound for use in asubunit immunogenic compositions.

[0126] In another embodiment, expressed Csa proteins or fragmentsthereof, or whole CS4 antigen complexes can be cross-linked to animmunogenic component. The immunogenic component can also be an isolatedprotein, functional fragment thereof, whole organism (such as abacterium or a virus), or functional fragment thereof, that is isolatedeither in part or as is used as a whole pathogenic organism. The Csaproteins can be isolated from the bacterium itself or it can be producedusing recombinant DNA techniques well known in the art.

[0127] In accordance with one aspect of the present invention, thesmaller fragments of expression product of the csa operon are used toprovide an immunogenic composition. Specifically, these fragments willcomprise an immunogenic region of such expression product, typicallyfrom about 5, 6, 8, 10 or 12 amino acids to about 20, 22, 24, 30, ormore amino acids. Suitable fragments or immunogenic regions can bereadily ascertained using as screening procedures the techniques setforth in Examples 1-4. In one embodiment of a suitable screeningprocedure, a large number of candidate fragments are more or lessrandomly produced and used to immunize guinea pigs or other suitablemodels. Alternatively, full-length polypeptides shown to be active inthe present invention can be truncated and screened in an iterativeprocess to isolate the immunogenic and protective activity to a minimalfragment. Such screening can be readily carried out without undueexperimentation and the active fragments are within the contemplation ofthe present invention.

[0128] The Csa proteins used to form the immunogenic compositions ofthis embodiment can be the whole protein, such as the CsaB or CsaEproteins, an immunogenic fragment of a Csa protein, a mutagenized formof a Csa protein, or a fusion protein comprising a Csa protein or afragment thereof and a suitable fusion partner. A suitable fusionpartner for such a Csa fusion protein or a fragment thereof might be anyprotein, peptide or amino acid sequence that facilitates the expressionand/or purification of the Csa fusion protein using recombinant DNAtechniques known in the art. Alternatively, one or more additionalimmunogens can serve as the fusion partner in a Csa fusion protein.

[0129] Nucleotide Immunogenic Compositions

[0130] In another embodiment, an immune response can be elicited usingnucleotide-containing compositions. In one aspect of this embodiment, amucosal or systemic immune response is elicited in a host byadministering an antigen-encoding polynucleotide preparation, comprisingDNA or RNA, which encodes an antigenic epitope to the host. In apreferred embodiment, the nucleotide-containing composition isadministered to a mucosal inductor site in the mucosal tissue of thehost.

[0131] Naked DNA may be administered directly to mucosa, for instance insaline drops, or in a recombinant gene expression vector. Preferably,the recombinant gene expression vectors are not capable of replicationor dissemination. Nucleotide-containing immunogenic compositions alsocomprise live viral immunogenic compositions wherein the viruses includeimmunostimulatory polynucleotides. According to a preferred method ofthe invention, a target protein antigen is administered through itsexpression by a recombinant gene expression vector.

[0132] U.S. Pat. No. 6,110,898, to Malone, et al., entitled, “DNAvaccines for eliciting a mucosal immune response,” which is herebyincorporated by reference in its entirety, provides detailed teachingfor the generation of such immunogenic compositions.

[0133] Formulations and Administration

[0134] The immunogenic compositions described herein can be formulatedin a variety of useful formats for administration by a variety ofroutes. Concentrations of the immunogenic components in the formulationsdescribed will be such that an effective dose of the immunogeniccomponents are included in the formulation. Determination of such aconcentration would be readily apparent to those of ordinary skill inthe art.

[0135] Administration of the immunogenic compositions can be by nasalapplication, by inhalation, ophthalmically, orally, rectally, vaginally,or by any other mode that results in the immunogenic contacting themucosal tissues.

[0136] In one embodiment, the immunogenic composition exists as anatomized dispersion for use in delivery by inhalation. The atomizeddispersion of the immunogenic components will typically contain carriersfor atomized or aerosolized dispersions, such as buffered saline andother compounds well known to those of skill in the art. The delivery ofthe described immunogenic compositions via inhalation has the effect ofrapid dispersion to a large area of mucosal tissues, as well asabsorption by the blood for circulation of the immunogenic components.One example of a method of preparing an atomized dispersion is found inU.S. Pat. No. 6,187,344, entitled, “Powdered Pharmaceutical FormulationsHaving Improved Dispersibility,” which is hereby incorporated byreference in its entirety.

[0137] The immunogenic compositions described herein can also beformulated in the form of a suppository, whether rectal or vaginal.Typical carriers for formulation of the inactive portion of asuppository include polyethylene glycol, glycerine, cocoa butter andother compounds well known to those of skill in the art. Othersuppository formulations suitable for delivery of the describedimmunogenic compositions are also contemplated. Delivery of thedescribed immunogenic compositions via suppository it hypothesized tohave the effect of contacting a mucosal surface with the immunogeniccompositions for release to proximal mucosal tissues. Distal mucosaltissues also receive the immunogenic compositions by diffusion.

[0138] Additionally, immunogenic compositions are contemplated asexisting in a liquid form. The liquid can be for oral dosage, forophthalmic or nasal dosage as drops, or for use as an enema or douche.When the immunogenic compositions is formulated as a liquid, the liquidcan be either a solution or a suspension of the immunogeniccompositions.

[0139] A colloidal dispersion system may be used for targeted deliveryof nucleic acid-containing immunogenic compositions. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. A preferredcolloidal system is a lipid preparation including unilamaller andmultilamellar liposomes.

[0140] Liposomes are artificial membrane vesicles that are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the polynucleotides at high efficiency while not compromisingtheir biological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988). In additionto such LUV structures, multilamellar and small unilamellar lipidpreparations that incorporate various cationic lipid amphiphiles canalso be mixed with anionic polynucleotides to form nucleolipidicparticles which are often also referred to as liposomes (Felgner, et al,Proc Natl. Acad. Sci. U.S.A. 84 (21): 7413 1987) and used to deliver thenucleic acids into cells.

[0141] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. The appropriate composition andpreparation of cationic lipid amphiphile:polynucleotide formulations areknown to those skilled in the art, and a number of references whichprovide this information are available (e.g., Bennett, et al, J.Liposome Res. 6(3):545).

[0142] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.Examples of cationic amphiphilic lipids useful in formulation ofnucleolipid particles for polynucleotide delivery include the monovalentlipids DOTAP, DOTMA, and DC-Chol, the polyvalent lipids LipofectAMINE,DOGS, Transfectam and other amphiphilic polyamines. These agents may beprepared with helper lipids (such as Dioleoyl Phosphatidyl Ethanolamine)or with various carrier compositions, including various adjuvants, suchas cholera-derived molecules including cholera toxin.

[0143] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs that contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0144] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0145] There are a variety of suitable formulations for the solution orsuspension well known to those of skill in the art, depending on theintended use thereof.

[0146] Delivery of the described immunogenic compositions in liquid formvia oral dosage has the aim of exposing the mucosa of thegastrointestinal and urogenital tracts to the immunogenic compositions.A suitable dose, stabilized to resist the pH extremes of the stomach,would deliver the immunogenic compositions to all parts of thegastrointestinal tract, especially the upper portions thereof. All meansof stabilizing the immunogenic compositions in a liquid oral dosage suchthat the effective delivery of the composition is distributed along thegastrointestinal tract are contemplated for use with the immunogeniccompositions described herein.

[0147] Delivery of the described immunogenic compositions in liquid formvia ophthalmic drops has the aim of exposing the mucosa of the eyes andassociated tissues to the immunogenic compositions. A typical liquidcarrier for eye drops is buffered and contains other compounds wellknown to those of skill in the art.

[0148] Delivery of the described immunogenic compositions in liquid formvia nasal drops has the aim of exposing the mucosa of the nose andsinuses and associated tissues to the immunogenic compositions. Liquidcarriers for nasal drops are typically various forms of buffered saline.

[0149] Administration of the compounds discussed above can be practicedin vitro or in vivo. When practiced in vitro, any sterile, non-toxicroute of administration may be used. When practiced in vivo,administration of the compounds discussed above may be achievedadvantageously by subcutaneous, intravenous, intramuscular, intraocular,oral, transmucosal, or transdermal routes, for example by injection orby means of a controlled release mechanism. Examples of controlledrelease mechanisms include polymers, gels, microspheres, liposomes,tablets, capsules, suppositories, pumps, syringes, ocular inserts,transdermal formulations, lotions, creams, transnasal sprays,hydrophilic gums, microcapsules, inhalants, and colloidal drug deliverysystems.

[0150] The compositions described herein are administered in apharmaceutically acceptable form and in substantially non-toxicquantities. A variety of forms of the compounds administered arecontemplated. The compounds may be administered in water with or withouta surfactant such as hydroxypropyl cellulose. Dispersions are alsocontemplated, such as those utilizing glycerol, liquid polyethyleneglycols, and oils. Antimicrobial compounds may also be added to thepreparations. Injectable preparations may include sterile aqueoussolutions or dispersions and powders, which may be diluted or suspendedin a sterile environment prior to use. Carriers such as solvents ordispersion media contain water, ethanol polyols, vegetable oils and thelike may also be added to the compounds described herein. Coatings suchas lecithins and surfactants may be used to maintain the proper fluidityof the composition. Isotonic agents such as sugars or sodium chloridemay be added, as well as products intended to delay absorption of theactive compounds such as aluminum monostearate and gelatin. Sterileinjectable solutions are prepared according to methods well known tothose of skill in the art and can be filtered prior to storage and/oruse. Sterile powders may be vacuum or freeze dried from a solution orsuspension. Sustained-release preparations and formulations are alsocontemplated. Any material used in the composition described hereinshould be pharmaceutically acceptable and substantially non-toxic in theamounts employed.

[0151] Although in some of the experiments that follow the compounds areused at a single concentration, it should be understood that in theclinical setting, the compounds may be administered in multiple dosesover prolonged periods of time. Typically, the compounds may beadministered for periods up to about one week, and even for extendedperiods longer than one month or one year. In some instances,administration of the compounds may be discontinued and then resumed ata later time.

[0152] All compound preparations may be provided in dosage unit formsfor uniform dosage and ease of administration. Each dosage unit formcontains a predetermined quantity of active ingredient calculated toproduce a desired effect in association with an amount ofpharmaceutically acceptable carrier. Such a dosage would thereforedefine an effective amount of a particular compound.

[0153] The immunogenic compositions described herein can be administeredin amounts appropriate to those individual compounds to produce animmune response. Appropriate doses for each can readily be determined bytechniques well known to those of ordinary skill in the art. Such adetermination will be based, in part, on the tolerability and efficacyof a particular dose using techniques similar to those used to determineproper chemotherapeutic doses.

[0154] Additionally, a kit comprising the necessary components of aimmunogenic composition that elicit an immune response to a selectedimmunogenic component are also contemplated.

EXAMPLE 1 Isolation and Characterization of the csa Operon

[0155] The csa operon that encodes the synthesis of ETEC-CS4 pili wasisolated from strain E11881A, cloned and sequenced. The csa operonconsist of 5 contiguous genes encoding CsaA-CsaE proteins, which sharehomology to other pili assembly proteins, and especially to CFA/I. Thecsa operon was expressed in an attenuated Shigella strain, CVD1204guaBA, constructing the Shigella expressing CS4 fimbriae vaccine strainCVD1204 (pGA2-CS4). Immunization of guinea-pigs with CVD1204 (pGA2-CS4)elicited the production of anti-CS4 antibodies that reacted with CS4producing strains and prevented biological activities mediated by ETECstrains. This work contributes to previously reported results regardingthe on expression of CFA/I, CS2, CS3 and LT in attenuated Shigella,emphasizing the feasibility of constructing an efficient multivalentShigella-based oral ETEC vaccine.

[0156] The genes that encode the synthesis of ETEC CS4 fimbriae,csaA,B,CD,E,D′, (the csa operon), were isolated from strain E11881A. Thecsa operon encodes a 17 kDa major fimbrial subunit (CsaB), a 40 kDa tipassociated protein (CsaE), a 27 kDa chaperon like protein (CsaA), a 97kDa usher protein (CsaC), and for a deleted regulatory protein (CsaD′)containing 100 amino acids out of 265. The csa operon is flanked by IS1Ethat inserted into the csaD′ gene, and IS21 sequences upstream to thecsaA. The csa operon is located on the large virulence plasmid thatcarries the LT genes.

[0157] A BLAST search of the predicted amino acid sequences indicatedhigh homology of the CS4 proteins to structural and assembly proteins ofCFA/I in particular, and to CS 1 and CS2 fimbriae proteins. ThecsaA,B,C,E operon was cloned on a 15 copy number stabilized plasmid downstream from an osmotically regulated ompC promoter. Plasmid pGA2-CS4directs in both DH5α and Shigella flexneri 2a strains the production ofCS4 fimbriae, as detected by western blot analysis and bacterialagglutination using anti-CS4 fimbriae immune sera. Electron microscopicexamination of Shigella expressing the CS4 fimbriae indicated theproduction of lots of rod like shape extensions. Immunization of guineapigs with S. flexneri 2a CVD1204 (pGA2-CS4) elicited the production ofanti-CS4 antibodies that bind to CS4 fimbrial proteins, agglutinates CS4producing strains, inhibits hemagglutination by ETEC strains andprevented the adhesion of ETEC strains to human mucosal cell lineCaco-2.

[0158] Isolation of the csa Operon

[0159] Genomic DNA of strain E 11881A was isolated using GNOME DNA KIT(BIO 101, Carlsbad, Calif.) protocol. The DNA was partially digestedwith Sau3AI, the DNA fragments with size of 5−>20 kilobase pairs wereisolated from agarose gels and ligated into vector pKS that was digestedwith BamHI and treated with shrimp alkaline phosphatase. The ligationmix was transformed into DH5α. DH5α transformants were grown in LB inthe presence of carbenicillin at 50 μg/ml or kanamycin at 50 μg/ml.

[0160] The resulting transformants were harvested into 96 wellmicrotiter plates, and assayed for the csa operon by PCR tests, based onthe published sequence of the csfA gene, (the structural gene of the CS4fimbriae, NCBI Accession number X97493). Based on that sequence, twoprimers were constructed which amplified 319 base pair fragment. Theprimers are: CS44 bp 1 to 29: GTTGACCCTACAATTGATATTTTGCAAGC (SEQ IDNO:11) CS45 bp 378 to 348: CGACCCCACTATAATTCCCGCCGTTGGTGC (SEQ IDNO:12). Pools of 1200 colonies were analyzed and 2 colonies werepositive in the PCR test. Both colonies were found to contain the sameplasmid, pKS-CSA-I, (FIG. 1A). Sequencing of the cloned DNA fragment,indicated that plasmid pKS-CSA-I contains most of the csa operon,missing 430 bp from the 5′ end of the csaA gene. DNA sequencing of thecsa operon revealed a high degree of homology to the DNA sequences ofthe CFA/I operon (Table 2).

[0161] Based on that homology, two new primers were designed forscreening the genomic library for the 5′ end by PCR. Primer CS433 isbased on the csaA sequences at bp 718, and primer CS434 is based on theCFA/I sequences from bp 878. CS433: GTGATATGTTTTGTTCACTTGGTAAAGATC (SEQID NO:13) CS434: CTCATGGCTCCATTTGTTGCAAATGCAAACTTTATG (SEQ ID NO:14).PCR assays using these primers amplified a 429 bp DNA fragment from thegenomic DNA of strain E 118811A. By screening the DNA library, apositive clone that contained the entire csaA gene together withupstream DNA sequences was isolated. The clone contains the 8000 basepair plasmid pKS-CSA-II, (FIG. 1B).

[0162] DNA Sequence Analysis

[0163] DNA sequences of the CS4 encoding genes in plasmids pKS-CSA-1 andpKS-CSA-II were determined in both strands. The sequencing primers weresynthesized on Perkin Elmer DNA Synthesizer model 3948 at the 40 nMsynthesis scale. The sequencing was performed using a Perkin Elmer DNASynthesizer 373, with the Dye Terminators from the BIGDYE KIT, PerkinElmer (Boston, Mass.). The sequencing results identified the csa operonand its flanking genes, as is schematically described in FIG. 1C.

[0164] Construction of pGA2

[0165] The vector used for expression of the CS4 encoding genes inShigella, pGA2, was derived from plasmid pEXO1 by replacing the gfpencoding gene (751 bp DNA, flanked by the restriction enzymes Clal andNheI), with an 87 bp linker that contains multiple cloning sites (mcs),that was constructed by PCR, as indicated. pEXO1 is an expressionplasmid derived from pGEN222. (Galen, et al., Infect. Immun.,67:6424-6433 (1999)). Plasmid pGEN222 carries a two-component plasmidmaintenance system comprised of the hok-sok post-segregational killingsystem plus the parA plasmid partitioning system. these two componentshave been shown to work in concert to minimize plasmid loss from apopulation of actively growing bacteria and to lyse any bacteria fromwhich plasmids have segregated. pEXO1 was created from pGEN222 byreplacing the bla gene, encoding β-lactamase, with an engineered aphallele encoding reduced levels of resistance to kanamycin (Blomfield, etal., Mol. Microbiol., 5:1447-1457 (1991)). It was observed thatexpression of the wildtype aph allele within attenuated S. typhi vaccinestrains lead to plasmid instability. Therefore, transcription of thisaph allele was modified by PCR such that the separation of the −35 and−10 regions within the promoter was increased from 18 to 19 base pairs;this re-engineered allele was then introduced as an Nhe I fragment intopGEN222 cleaved with Xba I and Spe I to replace the bla gene.

[0166] The PCR fragment encoding for mcs was used as a Cla I-Nhe Irestriction fragment to replace the Cla I-Nhe I restriction fragmentwithin pEXO1 encoding the gfpuv allele, creating pGA2. pGA2 is thereforeexpected to be present at approximately 15 copies per chromosomalequivalent, and to drive expression of CS4 encoding genes from theosmotically responsive ompC promoter.

[0167] The mcs was constructed by PCR using the following primers:Primer 4a: GGGATCGATCCCGGGGCGGCCGCGGGCCCGGTACCAGGCCTTCTAGAAAGC TTGACGTCG(SEQ ID NO: 15); primer 4b:CCCGCTAGCGGCGCGCCTCGCGAGGATCCGTCGACGACGTCAAGCTTTCT AGA AGGCCTGG (SEQ IDNO: 16); Primer 4c: AAGCTTGACGTCGTCGACGG (SEQ ID NO: 17); Primer 4d:CCCGCTAGC GGCGCGCCTCGCG (SEQ ID NO: 18).

[0168] Construction of the CS4 Encoding Plasmid

[0169] Plasmid pGA2-CS4 (FIG. 2B), was constructed in three steps. The5′ end of the csaA gene was cloned from plasmid pKS-CSA-II on a 710 bpHpaI/XbaI fragment, into pKS SmaI/XbaI. The HpaI site is located 241 bpupstream the ATG codon for the csaA gene. The DNA fragment was thenfurther cloned as a KpnI/XbaI fragment into pGA2 KpnI/XbaI, to constructpGA2-ΔcsaA plasmid. The remaining CS4 encoding genes, (the 3′ end ofcsaA, csaB, csaC, and csaE) were cloned from pKS-CSA-I as a 4526 bp XbaIfragment. The two XbaI sites are located in the csaA gene, and in thestop codon of the csaE gene. The DNA fragment was ligated to pGA2-ΔcsaAXhaI site, to construct plasmid pGA2-CS4.

[0170] Additional DNA Primers for PCR Assays

[0171] For location of different genes by PCR assays, the followingprimers were constructed. For LT gene (AB011677, GI 3062900): LTA bp162:CCGTGCTGACTCTACACCCCCAGATG (SEQ ID NO: 19) and LTB bp895:GCACATAGAGAGGATAGTAACGCCG (SEQ ID NO:20). For gyrA gene (X57174, GI41641): GYRA bp 347:CGGTCATTGTTGGCCGTGCGCTGCC (SEQ ID NO:21) and GYRA bp1147:CACGCAGCGCGCTGATGCCTTCCACGCG (SEQ ID NO:22). For csaD gene: CS4D3:from csa operon bp 5540-5564, and cfaD (GI 145505) bp 1236-1260.CATATTTGATATCTGAGATATCTGG (SEQ ID NO:23) CS4D2: From csa operon bp6005-60030, and cfaD bp 771-794, TGTTGCATTCAGATTGAACGGAG (SEQ ID NO:24).CS4D1: From cfaD bp 606-629, at similar region to rns. This DNA regionis missing in csa operon: TATTATGATTCATAAATACACTGT (SEQ ID NO:25). PCRassays using primers CS4D2/CS4D3 is expected to amplify a 476 bp DNA,and with primers CS4D1/CS4D3, a 646bp DNA.

[0172] Transformation of Shigella Strains

[0173] Shigella strains were grown in trypticase soy agar (TSA) agarplates containing 0.1% Congo red and 10 μg/ml guanine. Competent cellsof S. flexneri 2a CVD1204 were prepared by growing the cells in L brothsupplemented with guanine to OD_(600 nm) of 0.6. The cells wereprecipitated, washed twice with cold H₂O, once with cold 10% glyceroland resuspended in the same buffer at 1/100 of the original volume. Amixture of 150 μl cells and plasmid DNA were electroporated in a 0.2 cmcurettes in a Gene Pulser (BioRad Laboratories, Hercules, Calif.) using2.5 kV, 200 Ω, 25 μF, or 1.75 kV, 600 Ω, 25 μF. Transformants wereselected on kanamycin, guanine and Congo red containing TSA plates.

[0174] Detection of Pili Synthesis

[0175] ETEC strains were grown on CFA (Hamers, et al., Microb. Pathog.,6:297-309 (1989)) plates at 37° C., and the bacteria was resuspended inphosphate buffered saline (PBS). Shigella strains that contain theplasmid pGA2-CS4 were grown in TS broth, [Trypton (Difco), 1.5%; Soyton(Difco), 0.5%)], containing 0, 50, 150 and 300 mM NaCl to a logarithmicphase growth. The bacteria were assayed for pili production by bacterialagglutination assays, and by immunoblotting of cell extracts. Forimmunoblotting the bacterial cultures were adjusted to OD_(600 nm)=10and boiled for 10 minutes in Laemnli sample buffer (BioRad). The cellextracts proteins were separated on SDS-PAGE (15%), transferred tonitrocellulose filters (MSI) and probed with anti-CS4 serum. Thespecific anti-CS4 antiserum was produced in rabbits by immunization withETEC strain E11881A (a CS4⁺ CS6⁺ producer strain), and absorption of thesera on EII88IC (a CS4⁻CS6⁺ strain).

[0176] Hemagglutination

[0177] For hemagglutination tests (Willshaw, et al., FEMS Microbiol.Lett., 49:473-478 (1988)), ETEC and Shigella strains were grownovernight on CFA or TSA/CR/guanine /kanamycin containing plates,respectively, and were resuspended in PBS to OD_(600 nm) of 10. Theslide hemagglutination tests, were performed as described by Sakellaris,et al 1999 (Sakellaris, et al., Proc. Natl. Acad. Sci. U.S.A.,96:12828-12832 (1999)), by mixing 20 μl bacterial suspension with 20 μlPBS containing 0.1M D-mannose and 20 μl washed human erythrocytes ofgroup A. For hemagglutination inhibition assays, the bacterialsuspensions were incubated with four-fold diluted antibodies for 1 hourat 37° C. prior to the hemagglutination tests.

[0178] Adhesion and Inhibition of Adhesion to Caco-2 Cells

[0179] Caco-2 cells were grown for 15 days in 8 chamber slides inDulbecco's modified Eagels medium containing fetal calf serum (20%) andglutamine (1%). Adhesion assays were performed by incubating washedcells with bacterial concentrations of 10⁷ and 10⁸/ml, for 2 and 3 hoursin DMEM containing 0.1M D-mannose, (Viboud, et al., Microb. Pathog.,21:139-147 (1996)). In the inhibition of adhesion assays, the bacterialcultures were incubated with antibodies for 1 hour, at 37° C. prior totheir addition to the washed Caco-2 cells. Following the incubation, theCaco-2 cells were washed 5 times with PBS, fixed with methanol andstained with Giemsa (10%).

[0180] Immunization

[0181] Guinea pigs, anesthetized subcutaneously with ketamine HCL (40mg/kg) and xylazine (5 mg/kg), were inoculated intranasal administrationtwice on days 0 and 14, with ˜2×10⁹ bacteria (0.1 ml of 40OD₆₀₀ nm) thatgrew on TSA/Congo red/guanine containing plates and harvested in PBS.Two groups of animals were immunized: group I with CVD1204 (pGA2), andgroup II with CVD1204 (pGA2-CS4), a CS4 producing strain. Sera wereobtained on days 0, 14 and 30 by anterior vena cava puncture ofanesthetized animals.

[0182] Results

[0183] Cloning and Sequencing of the CS4 Fimbriae Encoding Genes

[0184] The csa operon that encode the synthesis of the CS4 fimbriae, wasisolated from a genomic DNA library of strain E11881A, as describedabove. Most of the csa operon was cloned on plasmid pKS-CSA-I thatcontains the carboxy terminal part of the csaA gene, the csaB, csaC,csaE genes and a disrupted csaD′ gene. The csaD′ was disrupted byintegration of an IS1 element, creating a deletion of the amino terminal48 amino acids, and a frame shift mutation that resulted in stop codon.Agglutinations assays of DH5α (pKS-CSA-I) strains with rabbit serum antiE11881A (that was absorbed on strain E11881C) were positive, whichindicated expression of the CS4 fimbriae. Western blot tests indicatedthe presence of two fimbrial bands, the mature 17 kDa protein, and ahigher MW protein, probably the pre cleaved form. By further screeningof the CS4 library plasmid pKS-CSA-II was isolated. The plasmid containsthe csa promoter site, the csaA gene and the amino terminal region ofcsaB. Up stream of the csaA gene is an IS 21 element. The csa operon islocated on a ˜10,500 bp DNA fragment that is flanked by insertionelements, similar to a pathogenicity island, as schematically describedin FIG. 1C.

[0185] 7,239 bp of the CS4 pathogenicity island was sequenced in bothdirections, indicating a 34.88% G+C region. The csa operon is locatedbetween bp 1 and 6,095 bp. It contains 5 ORF, 4 genes, csaA, csaB, csaC,and csaE are transcribed in the same direction down stream from apredicted promoter site, and csaD′ from the antiparallel strand. Thelocation of each gene is described in Table 1, and schematicallypresented in FIG. 1C. A BLAST search for homology of the csa DNAsequence (bases 1 to 6096) to other genes indicated homology to CFA/I,CS1 and CS2 ETEC fimbriae encoding genes, to the structural genes of CS4and CS14, and to fimbriae regulatory genes cfaD, rns, csvR and aggR, aspresented in Table 2. The results of the BLAST search indicate that thecsa operon has a high DNA similarity to cfaI operon; 5,420 bp out of6,069 bp are >91% similar. Comparing the DNA sequence of each gene toother fimbriae encoding genes indicate the following results:

[0186] csaA: 93% of the 717 bp of the csaA DNA sequences are identicalto 716 bp of cfaA (M55661.1), and 44 bp from its 5′ end are 90%identical to csoA and cooB 713 bp genes (X62879.1 and X62495.1).

[0187] csaB: 402 bp of csfA (X97493.1), the published DNA sequence ofthe CS4 structural gene, are 99% identical to 501 bp of the csaB DNAsequences. Of the 512 bp of cfaB, (M55661.1), the sequence of 107 bp is93% identical to csaB. 93 bases from the 5′ end of csuA1 504 bp,(X9749.1), the structural gene for CS14 fimbriae, are 89% identical tocsaB. 35 bp from the 5′ end of cotA 512 bp gene (Z47800.1) is 94%identical to csaB.

[0188] csaC: 2601 bp of csaC gene share 96% DNA sequences similarity to2507 bp of cfaC (M55661.1), 80% homology to 391 bp out of 2618 bp ofcooC (X76908.1), and 85% homology to 124 bp out of 2597 bp of cotC(Z47800.1) genes.

[0189] csaE: 1086 bp of the csaE DNA sequences are >84% identical to 999bp out of 1082 bp of cfaE (M55661.1), and 32 bp out of 1091 bp of cooD(X76908.1) are 90% identical to csaE.

[0190] csaD′: csaD′ DNA sequence is similar to cfaD′ (CFA/I), cfaD, rnsand csvR from ETEC strains, and aggR from EHEC fimbriae. DNA sequencesimilarity between csaD′, cfaD and ms is of 751/817 bp (91%) for cfaD,and 721/787 (91%) for rns.

[0191] Comparing the regions of similarity in both genes indicated thatcsaD′ gene is missing 143 bp from its 5′-end because of an insertion ofan IS element. Blast search for DNA sequences downstream the csa operonindicated that from base 6096 to 6870 there is homology to IS1Esequences, (NCBI X52537, identities=765/769, 99%), from bp 6892 to 7054,there is homology to EPEC strain plasmid pB171 (GI 6009376), between bp16240 and 16471. And from bp 7062-7239 there is homology to Shigellasonnei strain P9 plasmid colIb (GI 4512437) between bp 4895 and 5568.

[0192] Promoter Site

[0193] The promoter site for the csa operon was predicted using the“Promoter Prediction By Neural Network”, and it is proposed to belocated between bp 145 to 194, 89 bp upstream the ATG codon for csaAgene. The predicted promoter sequences is as follow:

[0194] TGTGGGTATTTGTTTGGACATCGCAGCATTAAATATAAAAATAGCACAGG (SEQ IDNO:26). The large underline “A” is the predicted transcription start,and the shaded nucleotides are the possible −10 and −35 sequences. ABLAST search indicated a 28/31 bp (90%) homology to the CFA/I fimbrialoperon, at between bp 687 to 717, 131 bp upstream to the cfaA gene.

[0195] CS4 pili structural and assemble proteins. The csa operon encodesthe synthesis of five proteins: CsaA, CsaB, CsaC, CsaE and CsaD. Thelocation of the genes and the size of the putative proteins aredescribed in Table 1. According to sequence similarity of the CsaA-Eproteins to other ETEC fimbriae proteins, the predicted functions of theCsaA-E proteins is as follow:

[0196] CsaA function as a periplasmic chaperon like protein. PSORTanalysis indicate that the putative protein is a bacterial periplasmicspace protein (certainty of 0.939). CsaB is the major pilin subunit.CsaC is a membrane usher protein. A PSORT analysis predicted an outermembrane location (certainty of 0.926). CsaE is assumed to be at thefimbriae tip. A PHDsec analysis (for the prediction of secondarystructure) indicated that the protein is a compact protein as a globulardomain. CsaD′ is a fimbriae regulatory protein. The CsaD′ proteincontains 100 amino acids from the carboxy terminal part of the protein,missing the first 48 amino acids (based on homology to cfaD gene fromCFA/I pili), because of an insertion of an IS1E element. Following the100 amino acid is a frame shift mutation that encodes for a stop codon.

[0197] Homology of the CsaA-E Proteins to Other Fimbriae Proteins

[0198] A BLAST search with the putative amino acid sequence of CsaA-Eproteins indicated homology to fimbriae proteins from ETEC strains andSalmonella typhi as described in Table 3. The amino acid sequence of thestructural and assemble proteins of the CS4 fimbria have similarity toETEC proteins producing the CFA/I, CS1 and CS2 fimbriae, and to theSalmonella fimbria. In addition, the CS4 structural proteins have aminoacid sequences similar to the structural proteins of CS 14, CS17, CS19and B. cepacia. The CS4 fimbriae structural protein CsaB and thetip-associated protein CsaE are responsible for the fimbrial structureand for the bacterial attachment to intestine cells. An alignment of theamino acids sequences of the fimbrial proteins that are similar to CsaBand CsaE is described in FIGS. 3 and 4. TABLE 3 Similarity in AAsequences between ETEC CsaA-E proteins and other fimbrial proteinsregion in region in CS4 #amino CS4 compared Gene protein Pili Proteinacids Identities proteins protein no. CsaA CFA/I CfaA 238 208/238  (87%)1-238  1-238 gi 145508 CS1 CooB 238 124/221  (56%) 1-219  1-219 gi 95719CS2 CotB 238 111/238  (46%) 2-238  2-238 gi 897726 TsaA¹ 236 60/194(30%) 17-207  22-204 gi 5640159 CS5 25.9KD 224 52/218 (23%) P33792 CsaBCS4 CsfA 134 109/134  (81%) 34-160   1-134 gi 1304302 CFA/I CfaB 17099/170 (58%) 1-167  1-170 GI 145509 CS14 CsuA1 168 95/164 (57%) 1-164 1-164 gi 1304304 CS1 CooA 171 85/168 (50%) 1-164  1-168 gi 78442 CS2CotA 170 76/167 (45%) 1-164  1-167 gi 897727 CS14 CsuA2 142 67/132 (50%)34-164   1-132 gi 1304306 CS19 CsdA 133 56/131 (42%) 34-164   1-130 gi1304300 CS17 CsbA 135 54/132 (40%) 34-164   1-132 gi 1304298 TsaB¹ 19158/169 (34%) 1-166 22-187 gi 5640160 Cable Cb1A² 184 50/150 (33%)20-167  13-159 gi 606843 CsaC CFA/I CfaC 869 800/868  (92%) 16-883  2-869 gi 2121079 CS1 CooC 872 531/841  (63%) 40-880  31-868 gi 488736CS2 CotC 866 469/839  (55%) 40-878  29-864 gi 897728 CS6 CssD 120/549 (21%) P53513 TsaC¹ 895 260/867  (29%) 42-872  25-882 gi 5381204 AtfC³843 111/491  (22%) 211-670  207-665  gi 1504107 Caf1A⁴ 833 100/511 (19%) 209-685  205-661  gi 3883097 CsaE CFA/I CfaE 360 268/361  (74%)2-362  1-360 gi 2121080 CS1 CooD 363 177/329  (59%) 41-362  39-363 gi488737 CS2 CotD 364 161/339  (47%) 29-362  28-364 gi 897729 TsaD¹ 35990/303 (29%) 71-361  79-358 gi 5640162 rfbF⁵ 187 19/66  (28%) 146-211 55-114 gi 48590 CsaD CFA/I CfaD 265 90/101 (89%) 9-109 49-149 gi 145506CFA/I CfaD 144 90/101 (89%) 9-104 49-144 gi 145508 RNS 265 89/101 (88%)9-104 49-149 gi 145512 CSVR 305 75/103 (72%) 9-104 49-151 gi 95726 AAF/IAGGR 265 57/101 (56%) 9-109 49-149 gi 420983

[0199] Localization of the csa Pperon in Strain E11881A

[0200] In order to identify the location of the csa operon, totalgenomic DNA was isolated from strain E11881A, and was subjected toagarose gel electrophoresis. The electrophoresis results indicated thepresence of 3 plasmids: a large plasmid located above the chromosomalband, and two smaller plasmids located under the chromosomal DNA band.The plasmids and the chromosomal bands were gel eluted and tested by PCRassays for the presence of the csa operon, using primers CS44/CS45 foramplification of the csaB gene. These results indicated that the csaoperon was located on the large plasmid. The location of the LT gene wasdetected by PCR assays using primers LTA162/LTB895 (for amplification ofa 708 bp DNA), indicating the LT encoding genes were located on thelarge plasmid. Amplification of the gyrA gene, using primersGYRA347/GYRA 1147 (for amplification of a 748 bp DNA) indicated achromosomal location. In order to see whether strain E11881A contained acomplete csaD gene, PCR assays were performed with primers that arehomologous to csaD′ gene (CS4D2 and CS4D3), and with primer CS4D 1 thatis homologous to cfaD and rns genes (CSD41/CSD43). The results indicatesthat strain E11881A does not contain the complete cfaD gene. PrimersCS4D2/CS4D3 amplified the expected 476 DNA fragment, while primersCS4D1/CS4D3, did not amplified an expected 646 bp. Strains EII881E,DS9-1H10407, and C91f, contained the complete gene.

[0201] Expression of the CS4 Pili in DH5α and CVD1204

[0202] The expression of the CS4 fimbriae was detected following cloningof 5281 bp of the csa operon in vector pGA2, to construct the plasmidpGA2-CS4, (FIG. 2B). The cloned csa operon contained 240 bp of thepromoter region up stream the ATG codon for csaA gene, the csaA, csaB,csaC and csaE genes. The csa operon was cloned downstream of the ompCpromoter, which is osmotically regulated. Plasmid pGA2-CS4 wastransferred to E.coli DH5α and Shigella CVD1204 strains. CS4 fimbriaeproduction by strains DH5α (pGA2-CS4) and CVD1204 (pGA2-CS4) strains wasdetected by agglutination assays using rabbit serum against the CS4fimbriae and western blot of cell extracts. Western blot resultsindicates that the cloned csaABCE gene cluster encodes for a 17 kDa bandthat correspond to the CS4 fimbriae.

[0203] Antibody Response of Guinea-pigs to CVD1204 (pGA2-CS4)

[0204] To test whether the cloned csa operon could induce an immuneresponse against ETEC-CS4, guinea pigs were immunized by two intranasaladministrations of 2×10⁹ live CVD1204 (pGA2-CS4), and as control withCVD1204 (pGA2) strain. The immunized animals were tested for bacterialagglutination, immunoblotting, and inhibition of hemagglutination andadherence of CS4 expressing strains to Caco-2 cells.

[0205] Agglutination Assays

[0206] Bacterial agglutination assays done with immune guinea pigsindicated that immunized animals with CVD1204 (pGA2-CS4) developedantibodies that agglutinates all CS4 producing strains, and especiallythe high fimbriae producing strains DS 9-1, DH5α(pGA2-CS4) andCVD1204(pGA2-CS4), as described in Table 4. The end point of serumdilution that agglutinates DS 9-1 and DH5α(pGA2-CS4) strains is >1:100and >1:1000, respectively. The control sera from animals immunized withCVD1204 (pGA2) strain, agglutinate CVD1204 strains. Bacterialagglutination assays using rabbit anti-CS4 antiserum were also performedand the data from these assays is presented in Table 5. TABLE 4Bacterial agglutination assays by rabbit anti CS4 antiserum End pointdilution of NaCl the antibody that result Induction Strain mM inbacterial agglutination factor CVD1204(pGA2-CS4) 0   1:400 50   1:800 2150   1:1600 4 300   1:800 2 CVD1204(pGA2) 0 <1:10 150 <1:10 none

[0207] TABLE 5 Bacterial agglutination assays by immune guinea pigsserum Intensity of bacterial agglutination by immune guinea pigs sera²against: Relevant CVD1204 CVD1204 Tested strain¹ phenotype (pGA2-CS4)(pGA2) E11881A CS4⁺ CS6⁺ + − E11881E CS4⁺ CS6⁺ + − EII881C CS4⁻ CS6⁺ − −E11881/G28 CS4⁻ CS6⁻ − − DS9-1 CS4⁺ CS6⁺ ++++ − DH5α(pGA2-CS4) CS4⁺ ++++− DH5α CS4⁻ − − CVD1204(pGA2-CS4) CS4⁺ ++++ + CVD1204 CS4⁺ + ++

[0208] Immunoblotting Assays

[0209] The immunized guinea pigs were tested in immunoblottingexperiments to bind to CS4 fimbriae proteins. The assays were performedby probing CS4 producing strains with the guinea pig immune serum andrabbit anti-CS4 serum. Cell extract were isolated from ETEC strainsE11881A and DS 9-1, that grew on CFA plates at 37° C. and 22° C. (acondition that suppresses fimbriae production), from DH5α (pGA2-CS4) andCVD1204 (pGA2-CS4) strains that grew in LB broth in the presence of Km.The immunoblotting results showed that guinea pigs serum from CS4immunized animals reacted with a 17 kDa band protein which is the CS4fimbrial structural protein. ETEC strains produced a 17 kDa band at 37°C. but not at 22° C. E.coli and Shigella strains that contains theplasmid pGA2-CS4 produced 17 kDa protein bands, which was not producedin the corresponding untransformed cells.

[0210] Inhibition of Hemagglutination

[0211] ETEC strains that produce the CS4 fimbriae cause a mannoseresistant agglutination of human red blood cells type A. The ability ofETEC-CS4 strains, DH5α, and a CVD1204 strain that contains the clonedCS4 encoding genes, were tested for hemagglutination. The results arepresented in Table 6. The hemagglutination assays indicated that all CS4pili producing strains caused hemagglutination. ETEC-CS4 strainsdemonstrate a more intense hemagglutination than strains expressing thecloned csa operon. Non CS4 producing strains did not caused anyhemagglutination. TABLE 6 Efficiency of bacterial inducedhemagglutination Tested strains Relevant phenotype HemagglutinationE11881A CS4⁺ CS6⁺ ++ E11881E CS4⁺ CS6⁺ +++ EII881C CS4⁻ CS6⁺ − DS 9-1(37° C.*) CS4⁺ CS6⁺ ++++ DS 9-1 (22° C.*) CS4⁻ − DH5α(pGA2-CS4) CS4⁺ +DH5α CS4⁻ − CVD1204(pGA2-CS4) CS4⁺ + CVD1204(pGA2) CS4⁻ −

[0212] The guinea pigs anti-CS4 antiserum was tested for inhibition ofhemagglutination. ETEC-CS4 strains were incubated with various antibodydilutions prior to their addition to the red blood cells suspension. Theresults that are presented in Table 7 indicates that the guinea pigsanti-CS4 antibodies inhibits the hemagglutination, while the controlimmune serum against the Shigella has no effect on the ETEC-CS4 inducedhemagglutination. TABLE 7 Efficiency of inhibition of bacterial inducedhemagglutination by guinea pigs anti-CS4 antibodies End point dilutionof antibodies that inhibit bacterial Tested induced hemagglutination.Antibodies were from strains guinea pigs immunized with: E11881A 1:256<1:2 DS 9-1 1:32  <1:2

[0213] Inhibition of Adherence of ETEC-CS4 Strains to Caco 2 Cells

[0214] ETEC-CS4 producing strains have the ability to adhere to humancarcinoma cell line Caco-2. The adhesion of ETEC strain DS 9-1 to theCaco-2 cells, and the effect of the guinea-pigs anti-CS4 antibodies onthis adhesion were tested. The results indicate that ˜10-100 bacteriaadhere to the Caco-2 cells. As a control, the adhesion of DH5α bacteriawas tested, and only very few bacteria were found to adhere.Preincubation of the ETEC DS 9-1 with guinea pigs anti-CS4 serum (1:10dilutions), totally inhibit the adherence of the bacterial cells to theCaco-2 cells.

EXAMPLE 2 Construction of an Attenuated S. typhi Strain thatConstitutively Expresses CS4

[0215] In order to change the expression of CS4 in pGA2-CS4 fromosmotically regulated to constitutive, a strong promoter, e.g., P_(tac),is used. The promoter P_(tac) is constitutively active in Salmonellaspp., as these organisms lack laqI. Accordingly, constitutiveCS4-expressor derivatives of CVD 915 are constructed in the mannerdescribed below.

[0216] First, the ompC promoter is removed from the pGA2-CS4 constructand replaced with the P_(tac) promoter, resulting in the creation ofpGA2tac-CS4. After confirming the presence of the P_(tac) promoter inpGA2tac-CS4, the construct is introduced into S. typhi strain CVD-915,using standard molecular biology techniques.

[0217] Constitutive expression of the CS4 antigen is assessed by theagglutination assay described in Example 1. Using wild-type S. typhistrain Ty2 as the positive control, and untransformed CVD-915 (CVD 915⁻)as the negative control, expression of the CS4 antigen is found to bestrong, constitutive, and not regulated by changes in osmolarity.

EXAMPLE 3 Immune Response Against Constitutively Expressed CS4

[0218] Groups of ten 6 weeks old Balb/c mice are immunized intranasallywith 1.0×10¹⁰ cfu of strain CVD 915 with pGA2tac-CS4 (CVD 915⁺) or CVD915 without pGA2tac-CS4 (CVD 915⁻¹). Mice are bled prior and 30 daysafter their immunization, and their serum is stored at −20° C. untiluse. Antibodies present in the serum against S. typhi LPS, H (flagella)and CS4 antigens are determined by ELISA. The results indicate thatimmunization with strain CVD 915⁺ elicits antibody levels against theCS4 antigen that are significantly higher than those obtained withstrain CVD 915⁻. The immune responses against other S. typhi antigens(LPS and H) are similar between both immunized groups. The resultsdemonstrate that the constitutive expression of the CS4 antigen enhancesthe immune response against this antigen without interfering with theimmune response against other somatic S. typhi antigens.

EXAMPLE 4 Immune Response Against Constitutively Expressed CS4

[0219] The enterotoxic E. coli strain expressing the CS4 antigen(ETEC-CS4) from Example 1 is formulated into an immunogenic compositionfor nasal administration into a human subject (ETEC-CS4). Approximately1.0×10¹⁰ cfu of the recombinant bacteria ETEC-CS4 is nasallyadministered to a human subject. A booster is administered two weekssubsequent to the first administration. Blood is subsequently drawn fromthe subject and assayed for the presence of anti-ETEC-CS4 and anti-CS4antibodies. Such antibodies are found in the blood sample.

[0220] Although the invention has been described with reference toembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. All references cited herein are hereby expressly incorporated byreference.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 40 <210> SEQ ID NO 1<211> LENGTH: 717 <212> TYPE: DNA <213> ORGANISM: E. coli <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(717) <400> SEQUENCE: 1 atgcat aaa tta ttt tgt tta cta agt tta ctc ata act cca ttt gtt 48 Met HisLys Leu Phe Cys Leu Leu Ser Leu Leu Ile Thr Pro Phe Val 1 5 10 15 gcaaat gca aac ttt atg ata tat cca ata tca aaa gat tta aag aat 96 Ala AsnAla Asn Phe Met Ile Tyr Pro Ile Ser Lys Asp Leu Lys Asn 20 25 30 gga aatagc gag tta att cgt gtt tat tca aaa tca aaa gag ata caa 144 Gly Asn SerGlu Leu Ile Arg Val Tyr Ser Lys Ser Lys Glu Ile Gln 35 40 45 tat ata aaaata tat aca aaa aag att att aat ccc ggc aca act gaa 192 Tyr Ile Lys IleTyr Thr Lys Lys Ile Ile Asn Pro Gly Thr Thr Glu 50 55 60 gaa cat gaa gttgat atg ccc aat tgg gat ggt ggg ttt gta gtt act 240 Glu His Glu Val AspMet Pro Asn Trp Asp Gly Gly Phe Val Val Thr 65 70 75 80 cct caa aaa gttatt ctt cct gca gga ggg agt aaa tca ata cgt tta 288 Pro Gln Lys Val IleLeu Pro Ala Gly Gly Ser Lys Ser Ile Arg Leu 85 90 95 act caa ttt aga atacca aaa aaa gag gaa att tat aga gta tat ttt 336 Thr Gln Phe Arg Ile ProLys Lys Glu Glu Ile Tyr Arg Val Tyr Phe 100 105 110 gag gcg gta aaa ccagat agc aaa gaa aat gta att gat aat aaa aaa 384 Glu Ala Val Lys Pro AspSer Lys Glu Asn Val Ile Asp Asn Lys Lys 115 120 125 cta aca aca gag ctatct gtt aat ata att tat gcg gct cta atc aga 432 Leu Thr Thr Glu Leu SerVal Asn Ile Ile Tyr Ala Ala Leu Ile Arg 130 135 140 tct tta cca agt gaacaa aac ata tca cta aac att tct aga aat gca 480 Ser Leu Pro Ser Glu GlnAsn Ile Ser Leu Asn Ile Ser Arg Asn Ala 145 150 155 160 aga aaa aat ataatt att tat aat aat ggg aat gtt aga gca ggt gtt 528 Arg Lys Asn Ile IleIle Tyr Asn Asn Gly Asn Val Arg Ala Gly Val 165 170 175 aaa gat att tatttt tgt aag tca tct aat atc gat gat agc tgt gta 576 Lys Asp Ile Tyr PheCys Lys Ser Ser Asn Ile Asp Asp Ser Cys Val 180 185 190 aaa aaa acg cataac aag aat ata tat cca gaa aag tca ttt gat acg 624 Lys Lys Thr His AsnLys Asn Ile Tyr Pro Glu Lys Ser Phe Asp Thr 195 200 205 ctg gtt aat aacaat ttt tct tat gtt ttc att aaa tta aac cat gaa 672 Leu Val Asn Asn AsnPhe Ser Tyr Val Phe Ile Lys Leu Asn His Glu 210 215 220 gac ata gaa aaagag caa gga cta ata caa tta aaa gtt cct tga 717 Asp Ile Glu Lys Glu GlnGly Leu Ile Gln Leu Lys Val Pro * 225 230 235 <210> SEQ ID NO 2 <211>LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: E. coli <400> SEQUENCE: 2Met His Lys Leu Phe Cys Leu Leu Ser Leu Leu Ile Thr Pro Phe Val 1 5 1015 Ala Asn Ala Asn Phe Met Ile Tyr Pro Ile Ser Lys Asp Leu Lys Asn 20 2530 Gly Asn Ser Glu Leu Ile Arg Val Tyr Ser Lys Ser Lys Glu Ile Gln 35 4045 Tyr Ile Lys Ile Tyr Thr Lys Lys Ile Ile Asn Pro Gly Thr Thr Glu 50 5560 Glu His Glu Val Asp Met Pro Asn Trp Asp Gly Gly Phe Val Val Thr 65 7075 80 Pro Gln Lys Val Ile Leu Pro Ala Gly Gly Ser Lys Ser Ile Arg Leu 8590 95 Thr Gln Phe Arg Ile Pro Lys Lys Glu Glu Ile Tyr Arg Val Tyr Phe100 105 110 Glu Ala Val Lys Pro Asp Ser Lys Glu Asn Val Ile Asp Asn LysLys 115 120 125 Leu Thr Thr Glu Leu Ser Val Asn Ile Ile Tyr Ala Ala LeuIle Arg 130 135 140 Ser Leu Pro Ser Glu Gln Asn Ile Ser Leu Asn Ile SerArg Asn Ala 145 150 155 160 Arg Lys Asn Ile Ile Ile Tyr Asn Asn Gly AsnVal Arg Ala Gly Val 165 170 175 Lys Asp Ile Tyr Phe Cys Lys Ser Ser AsnIle Asp Asp Ser Cys Val 180 185 190 Lys Lys Thr His Asn Lys Asn Ile TyrPro Glu Lys Ser Phe Asp Thr 195 200 205 Leu Val Asn Asn Asn Phe Ser TyrVal Phe Ile Lys Leu Asn His Glu 210 215 220 Asp Ile Glu Lys Glu Gln GlyLeu Ile Gln Leu Lys Val Pro 225 230 235 <210> SEQ ID NO 3 <211> LENGTH:504 <212> TYPE: DNA <213> ORGANISM: E. coli <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(504) <400> SEQUENCE: 3 atg aaa ttaaaa aaa act att ggt gca atg gca ctg acc aca atg ttt 48 Met Lys Leu LysLys Thr Ile Gly Ala Met Ala Leu Thr Thr Met Phe 1 5 10 15 gta gct atgagt gct tct gca gta gag aaa aat atc act gta aca gct 96 Val Ala Met SerAla Ser Ala Val Glu Lys Asn Ile Thr Val Thr Ala 20 25 30 agt gtt gat cctaca att gat att ttg caa gct gat ggt agt agt tta 144 Ser Val Asp Pro ThrIle Asp Ile Leu Gln Ala Asp Gly Ser Ser Leu 35 40 45 cct act gct gta gaatta acc tat tca cct gcg gca agt cgt ttt gaa 192 Pro Thr Ala Val Glu LeuThr Tyr Ser Pro Ala Ala Ser Arg Phe Glu 50 55 60 aat tat aaa atc gca actaaa gtt cat aca aat gtt ata aat aaa aat 240 Asn Tyr Lys Ile Ala Thr LysVal His Thr Asn Val Ile Asn Lys Asn 65 70 75 80 gta cta gtt aag ctt gtaaat gat cca aaa ctt aca aat gtt ttg gat 288 Val Leu Val Lys Leu Val AsnAsp Pro Lys Leu Thr Asn Val Leu Asp 85 90 95 tct aca aaa caa ctc ccc attact gta tca tat gga gga aag act cta 336 Ser Thr Lys Gln Leu Pro Ile ThrVal Ser Tyr Gly Gly Lys Thr Leu 100 105 110 tca acc gca gat gtg act tttgaa cct gca gaa tta aat ttt gga acg 384 Ser Thr Ala Asp Val Thr Phe GluPro Ala Glu Leu Asn Phe Gly Thr 115 120 125 tca ggt gta act ggt gta tcttct tcc caa gat tta gtg att ggt gcg 432 Ser Gly Val Thr Gly Val Ser SerSer Gln Asp Leu Val Ile Gly Ala 130 135 140 act aca gca caa gca cca acggcg gga aat tat agt ggg gtc gtt tct 480 Thr Thr Ala Gln Ala Pro Thr AlaGly Asn Tyr Ser Gly Val Val Ser 145 150 155 160 atc tta atg acc tta gcatca taa 504 Ile Leu Met Thr Leu Ala Ser * 165 <210> SEQ ID NO 4 <211>LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: E. coli <400> SEQUENCE: 4Met Lys Leu Lys Lys Thr Ile Gly Ala Met Ala Leu Thr Thr Met Phe 1 5 1015 Val Ala Met Ser Ala Ser Ala Val Glu Lys Asn Ile Thr Val Thr Ala 20 2530 Ser Val Asp Pro Thr Ile Asp Ile Leu Gln Ala Asp Gly Ser Ser Leu 35 4045 Pro Thr Ala Val Glu Leu Thr Tyr Ser Pro Ala Ala Ser Arg Phe Glu 50 5560 Asn Tyr Lys Ile Ala Thr Lys Val His Thr Asn Val Ile Asn Lys Asn 65 7075 80 Val Leu Val Lys Leu Val Asn Asp Pro Lys Leu Thr Asn Val Leu Asp 8590 95 Ser Thr Lys Gln Leu Pro Ile Thr Val Ser Tyr Gly Gly Lys Thr Leu100 105 110 Ser Thr Ala Asp Val Thr Phe Glu Pro Ala Glu Leu Asn Phe GlyThr 115 120 125 Ser Gly Val Thr Gly Val Ser Ser Ser Gln Asp Leu Val IleGly Ala 130 135 140 Thr Thr Ala Gln Ala Pro Thr Ala Gly Asn Tyr Ser GlyVal Val Ser 145 150 155 160 Ile Leu Met Thr Leu Ala Ser 165 <210> SEQ IDNO 5 <211> LENGTH: 2604 <212> TYPE: DNA <213> ORGANISM: E. coli <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2604) <400>SEQUENCE: 5 atg aca aaa aaa aat aca tta tat ata acg atc atc gca atg ctaact 48 Met Thr Lys Lys Asn Thr Leu Tyr Ile Thr Ile Ile Ala Met Leu Thr 15 10 15 cca tat tca gtt ttt tcc gga gat ata ccc aac tct ttc cgt gat tta96 Pro Tyr Ser Val Phe Ser Gly Asp Ile Pro Asn Ser Phe Arg Asp Leu 20 2530 tgg gga gaa caa gat gaa ttt tat gaa gta aaa cta tat gga caa act 144Trp Gly Glu Gln Asp Glu Phe Tyr Glu Val Lys Leu Tyr Gly Gln Thr 35 40 45cta gga ata cat cga att aaa aca acc cca aca cat att aag ttt tat 192 LeuGly Ile His Arg Ile Lys Thr Thr Pro Thr His Ile Lys Phe Tyr 50 55 60 tcaccc gaa agc att tta gat aaa ata aat gta aaa aaa gaa aag gaa 240 Ser ProGlu Ser Ile Leu Asp Lys Ile Asn Val Lys Lys Glu Lys Glu 65 70 75 80 aagaaa ttg agt gtt ttg ttc act aat tct ttt tca aga aat ggc aat 288 Lys LysLeu Ser Val Leu Phe Thr Asn Ser Phe Ser Arg Asn Gly Asn 85 90 95 atg agttgt cag ggg aat gct act ata cag tat aac tgc aat tac att 336 Met Ser CysGln Gly Asn Ala Thr Ile Gln Tyr Asn Cys Asn Tyr Ile 100 105 110 aaa acaaaa tca gta gat gtc atc gtt gat gat gtt gat aat gtt gtt 384 Lys Thr LysSer Val Asp Val Ile Val Asp Asp Val Asp Asn Val Val 115 120 125 aac cttttt ata ggt aat gaa ttt ctg gat tct gaa gca cac aat gat 432 Asn Leu PheIle Gly Asn Glu Phe Leu Asp Ser Glu Ala His Asn Asp 130 135 140 gaa tatcat caa tta tca cga aat gta aaa aaa gct ttt ata caa agc 480 Glu Tyr HisGln Leu Ser Arg Asn Val Lys Lys Ala Phe Ile Gln Ser 145 150 155 160 cagaca att aat gtc tca gat tct ggg aag tat aaa agt ttg tct gtt 528 Gln ThrIle Asn Val Ser Asp Ser Gly Lys Tyr Lys Ser Leu Ser Val 165 170 175 tcaggg aat agc gcg ctg ggt att aca gat aca agt tat gct gtc tta 576 Ser GlyAsn Ser Ala Leu Gly Ile Thr Asp Thr Ser Tyr Ala Val Leu 180 185 190 aattgg tgg atg aat tac aat aaa ttt aat ggt tac agc aac aac gaa 624 Asn TrpTrp Met Asn Tyr Asn Lys Phe Asn Gly Tyr Ser Asn Asn Glu 195 200 205 agaaca atc aat agt ttg tac ttt aga cat gat tta gat aag aga tat 672 Arg ThrIle Asn Ser Leu Tyr Phe Arg His Asp Leu Asp Lys Arg Tyr 210 215 220 tattat caa ttt gga cga atg gat cgt aca gat ttg tca caa agt att 720 Tyr TyrGln Phe Gly Arg Met Asp Arg Thr Asp Leu Ser Gln Ser Ile 225 230 235 240agc ggg aac ttt aat ttt aac tta ctt cct tta ccc gat att gat ggt 768 SerGly Asn Phe Asn Phe Asn Leu Leu Pro Leu Pro Asp Ile Asp Gly 245 250 255ata agg aca gga acc aca caa tct tat atc aaa aat aca gat aag ttt 816 IleArg Thr Gly Thr Thr Gln Ser Tyr Ile Lys Asn Thr Asp Lys Phe 260 265 270atc gca tcc cct gta act gtt atg tta act aat ttt tcc aga gtg gaa 864 IleAla Ser Pro Val Thr Val Met Leu Thr Asn Phe Ser Arg Val Glu 275 280 285gct ttt cgc aat aat caa tta ttg ggc gta tgg tat tta gat tct gga 912 AlaPhe Arg Asn Asn Gln Leu Leu Gly Val Trp Tyr Leu Asp Ser Gly 290 295 300gta aat gaa tta gat aca gct cgt tta cct tat ggt agt tac gat ctt 960 ValAsn Glu Leu Asp Thr Ala Arg Leu Pro Tyr Gly Ser Tyr Asp Leu 305 310 315320 aaa tta aaa att ttt gaa aat act cag tta gtt cgt gaa gaa ata att 1008Lys Leu Lys Ile Phe Glu Asn Thr Gln Leu Val Arg Glu Glu Ile Ile 325 330335 cct ttt aat aaa ggg aga agt tct att ggt gat atg caa tgg gac gtt 1056Pro Phe Asn Lys Gly Arg Ser Ser Ile Gly Asp Met Gln Trp Asp Val 340 345350 ttc att cag gga ggg aat att att aat gac aag gat cgt tac ata gaa 1104Phe Ile Gln Gly Gly Asn Ile Ile Asn Asp Lys Asp Arg Tyr Ile Glu 355 360365 aaa caa aat aat cat aag tca tca gtt aat gct ggg cta cgt tta cca 1152Lys Gln Asn Asn His Lys Ser Ser Val Asn Ala Gly Leu Arg Leu Pro 370 375380 att acg aaa aat atc tct gtt caa caa gga gca tct gtt ata gat aat 1200Ile Thr Lys Asn Ile Ser Val Gln Gln Gly Ala Ser Val Ile Asp Asn 385 390395 400 aaa aat tat tat gaa ggg agt ctc aaa tgg aat tcc ggc att ctg tct1248 Lys Asn Tyr Tyr Glu Gly Ser Leu Lys Trp Asn Ser Gly Ile Leu Ser 405410 415 ggc tca cta aat agt gag ttc agt ttt ctt tgg gga gat aat gca aaa1296 Gly Ser Leu Asn Ser Glu Phe Ser Phe Leu Trp Gly Asp Asn Ala Lys 420425 430 ggt aat tat caa agt atc tcg tat acc gat gga ttt agt tta tca ttt1344 Gly Asn Tyr Gln Ser Ile Ser Tyr Thr Asp Gly Phe Ser Leu Ser Phe 435440 445 tat cat aat gat aag cgg gtc gat aat tgt gga aga aat tac aat gct1392 Tyr His Asn Asp Lys Arg Val Asp Asn Cys Gly Arg Asn Tyr Asn Ala 450455 460 ggt tgg agt gga tgc tac gaa tca tat tcg gca tct tta agt att cct1440 Gly Trp Ser Gly Cys Tyr Glu Ser Tyr Ser Ala Ser Leu Ser Ile Pro 465470 475 480 tta ttg gga tgg aca agt act ctg gga tat agt gac act tat agtgaa 1488 Leu Leu Gly Trp Thr Ser Thr Leu Gly Tyr Ser Asp Thr Tyr Ser Glu485 490 495 tca gtt tat aaa aac cat att ctt tct gaa tat ggt ttt tat aatcaa 1536 Ser Val Tyr Lys Asn His Ile Leu Ser Glu Tyr Gly Phe Tyr Asn Gln500 505 510 aac ata tat aaa ggg aga acc caa aga tgg caa ctg act tcg tccacc 1584 Asn Ile Tyr Lys Gly Arg Thr Gln Arg Trp Gln Leu Thr Ser Ser Thr515 520 525 tct tta aaa tgg atg gat tat aat ttt atg cca gca att gga atatat 1632 Ser Leu Lys Trp Met Asp Tyr Asn Phe Met Pro Ala Ile Gly Ile Tyr530 535 540 aac agt gag caa aga caa ctg act gat aaa ggc gga tat ata tctgta 1680 Asn Ser Glu Gln Arg Gln Leu Thr Asp Lys Gly Gly Tyr Ile Ser Val545 550 555 560 act ctc acc cga gcc agc aga gaa aat tca tta aac gca gggtat tct 1728 Thr Leu Thr Arg Ala Ser Arg Glu Asn Ser Leu Asn Ala Gly TyrSer 565 570 575 tac aac tat tcc aga gga aag tat tct tct aac gaa tta tttgtt gat 1776 Tyr Asn Tyr Ser Arg Gly Lys Tyr Ser Ser Asn Glu Leu Phe ValAsp 580 585 590 gga tat atg aca tca aca aat aat ggt gac tat cat gag gtaaga atg 1824 Gly Tyr Met Thr Ser Thr Asn Asn Gly Asp Tyr His Glu Val ArgMet 595 600 605 cgt ttt aat aaa aat aga cat aat gca gaa ggt aga ctt tcaggt cgt 1872 Arg Phe Asn Lys Asn Arg His Asn Ala Glu Gly Arg Leu Ser GlyArg 610 615 620 ata aac aat cga ttt gga gat tta aat ggt tca ttc agc atgaat aaa 1920 Ile Asn Asn Arg Phe Gly Asp Leu Asn Gly Ser Phe Ser Met AsnLys 625 630 635 640 aac aga aac acc aac agt agc aat cat tct ctc act ggtggt tat aat 1968 Asn Arg Asn Thr Asn Ser Ser Asn His Ser Leu Thr Gly GlyTyr Asn 645 650 655 tcc tca ttt gct ctt aca agt gat gga ttt tac tgg ggagga agt gca 2016 Ser Ser Phe Ala Leu Thr Ser Asp Gly Phe Tyr Trp Gly GlySer Ala 660 665 670 tct ggt ttg aca aaa cta gct ggc ggt att atc aag gttaaa tca aac 2064 Ser Gly Leu Thr Lys Leu Ala Gly Gly Ile Ile Lys Val LysSer Asn 675 680 685 gat act aaa aaa aat ctg gta aaa gtg act ggg gca ttgtac ggt gat 2112 Asp Thr Lys Lys Asn Leu Val Lys Val Thr Gly Ala Leu TyrGly Asp 690 695 700 tat tcg cta ggg agc aac gat aat gct ttt att cct gtacca gca tta 2160 Tyr Ser Leu Gly Ser Asn Asp Asn Ala Phe Ile Pro Val ProAla Leu 705 710 715 720 act cca gcc agt tta att att gaa gat aat aat tatggt gac aag aat 2208 Thr Pro Ala Ser Leu Ile Ile Glu Asp Asn Asn Tyr GlyAsp Lys Asn 725 730 735 att tct gta ctt gca cca acg aac aac gat atg tttata ttg ccg ggt 2256 Ile Ser Val Leu Ala Pro Thr Asn Asn Asp Met Phe IleLeu Pro Gly 740 745 750 aat gtt tat cct gtt gaa att gaa acc aaa gta agtgtt tct tat att 2304 Asn Val Tyr Pro Val Glu Ile Glu Thr Lys Val Ser ValSer Tyr Ile 755 760 765 ggt aga ggt ttt gac aaa aac ggc acg cca ctt tctggc gca cat gtt 2352 Gly Arg Gly Phe Asp Lys Asn Gly Thr Pro Leu Ser GlyAla His Val 770 775 780 ttg aat gaa cca cat gtt atc ctg gat gag gac ggtgga ttt tcg ttt 2400 Leu Asn Glu Pro His Val Ile Leu Asp Glu Asp Gly GlyPhe Ser Phe 785 790 795 800 gaa tat aca ggt aat gag aaa aca ctt ttt ttatta aag ggc aga act 2448 Glu Tyr Thr Gly Asn Glu Lys Thr Leu Phe Leu LeuLys Gly Arg Thr 805 810 815 att tat aca tgt caa ctg ggg aaa aat aaa gttcac aaa ggc att gtt 2496 Ile Tyr Thr Cys Gln Leu Gly Lys Asn Lys Val HisLys Gly Ile Val 820 825 830 ttc gtc gga gat gtt ata tgt gat gtt aat agcaca agt tcc tta cca 2544 Phe Val Gly Asp Val Ile Cys Asp Val Asn Ser ThrSer Ser Leu Pro 835 840 845 gat gaa ttt gta aag aac cca cgt gtg cag gatttg ctg gca aag aat 2592 Asp Glu Phe Val Lys Asn Pro Arg Val Gln Asp LeuLeu Ala Lys Asn 850 855 860 gat aaa gga taa 2604 Asp Lys Gly * 865 <210>SEQ ID NO 6 <211> LENGTH: 867 <212> TYPE: PRT <213> ORGANISM: E. coli<400> SEQUENCE: 6 Met Thr Lys Lys Asn Thr Leu Tyr Ile Thr Ile Ile AlaMet Leu Thr 1 5 10 15 Pro Tyr Ser Val Phe Ser Gly Asp Ile Pro Asn SerPhe Arg Asp Leu 20 25 30 Trp Gly Glu Gln Asp Glu Phe Tyr Glu Val Lys LeuTyr Gly Gln Thr 35 40 45 Leu Gly Ile His Arg Ile Lys Thr Thr Pro Thr HisIle Lys Phe Tyr 50 55 60 Ser Pro Glu Ser Ile Leu Asp Lys Ile Asn Val LysLys Glu Lys Glu 65 70 75 80 Lys Lys Leu Ser Val Leu Phe Thr Asn Ser PheSer Arg Asn Gly Asn 85 90 95 Met Ser Cys Gln Gly Asn Ala Thr Ile Gln TyrAsn Cys Asn Tyr Ile 100 105 110 Lys Thr Lys Ser Val Asp Val Ile Val AspAsp Val Asp Asn Val Val 115 120 125 Asn Leu Phe Ile Gly Asn Glu Phe LeuAsp Ser Glu Ala His Asn Asp 130 135 140 Glu Tyr His Gln Leu Ser Arg AsnVal Lys Lys Ala Phe Ile Gln Ser 145 150 155 160 Gln Thr Ile Asn Val SerAsp Ser Gly Lys Tyr Lys Ser Leu Ser Val 165 170 175 Ser Gly Asn Ser AlaLeu Gly Ile Thr Asp Thr Ser Tyr Ala Val Leu 180 185 190 Asn Trp Trp MetAsn Tyr Asn Lys Phe Asn Gly Tyr Ser Asn Asn Glu 195 200 205 Arg Thr IleAsn Ser Leu Tyr Phe Arg His Asp Leu Asp Lys Arg Tyr 210 215 220 Tyr TyrGln Phe Gly Arg Met Asp Arg Thr Asp Leu Ser Gln Ser Ile 225 230 235 240Ser Gly Asn Phe Asn Phe Asn Leu Leu Pro Leu Pro Asp Ile Asp Gly 245 250255 Ile Arg Thr Gly Thr Thr Gln Ser Tyr Ile Lys Asn Thr Asp Lys Phe 260265 270 Ile Ala Ser Pro Val Thr Val Met Leu Thr Asn Phe Ser Arg Val Glu275 280 285 Ala Phe Arg Asn Asn Gln Leu Leu Gly Val Trp Tyr Leu Asp SerGly 290 295 300 Val Asn Glu Leu Asp Thr Ala Arg Leu Pro Tyr Gly Ser TyrAsp Leu 305 310 315 320 Lys Leu Lys Ile Phe Glu Asn Thr Gln Leu Val ArgGlu Glu Ile Ile 325 330 335 Pro Phe Asn Lys Gly Arg Ser Ser Ile Gly AspMet Gln Trp Asp Val 340 345 350 Phe Ile Gln Gly Gly Asn Ile Ile Asn AspLys Asp Arg Tyr Ile Glu 355 360 365 Lys Gln Asn Asn His Lys Ser Ser ValAsn Ala Gly Leu Arg Leu Pro 370 375 380 Ile Thr Lys Asn Ile Ser Val GlnGln Gly Ala Ser Val Ile Asp Asn 385 390 395 400 Lys Asn Tyr Tyr Glu GlySer Leu Lys Trp Asn Ser Gly Ile Leu Ser 405 410 415 Gly Ser Leu Asn SerGlu Phe Ser Phe Leu Trp Gly Asp Asn Ala Lys 420 425 430 Gly Asn Tyr GlnSer Ile Ser Tyr Thr Asp Gly Phe Ser Leu Ser Phe 435 440 445 Tyr His AsnAsp Lys Arg Val Asp Asn Cys Gly Arg Asn Tyr Asn Ala 450 455 460 Gly TrpSer Gly Cys Tyr Glu Ser Tyr Ser Ala Ser Leu Ser Ile Pro 465 470 475 480Leu Leu Gly Trp Thr Ser Thr Leu Gly Tyr Ser Asp Thr Tyr Ser Glu 485 490495 Ser Val Tyr Lys Asn His Ile Leu Ser Glu Tyr Gly Phe Tyr Asn Gln 500505 510 Asn Ile Tyr Lys Gly Arg Thr Gln Arg Trp Gln Leu Thr Ser Ser Thr515 520 525 Ser Leu Lys Trp Met Asp Tyr Asn Phe Met Pro Ala Ile Gly IleTyr 530 535 540 Asn Ser Glu Gln Arg Gln Leu Thr Asp Lys Gly Gly Tyr IleSer Val 545 550 555 560 Thr Leu Thr Arg Ala Ser Arg Glu Asn Ser Leu AsnAla Gly Tyr Ser 565 570 575 Tyr Asn Tyr Ser Arg Gly Lys Tyr Ser Ser AsnGlu Leu Phe Val Asp 580 585 590 Gly Tyr Met Thr Ser Thr Asn Asn Gly AspTyr His Glu Val Arg Met 595 600 605 Arg Phe Asn Lys Asn Arg His Asn AlaGlu Gly Arg Leu Ser Gly Arg 610 615 620 Ile Asn Asn Arg Phe Gly Asp LeuAsn Gly Ser Phe Ser Met Asn Lys 625 630 635 640 Asn Arg Asn Thr Asn SerSer Asn His Ser Leu Thr Gly Gly Tyr Asn 645 650 655 Ser Ser Phe Ala LeuThr Ser Asp Gly Phe Tyr Trp Gly Gly Ser Ala 660 665 670 Ser Gly Leu ThrLys Leu Ala Gly Gly Ile Ile Lys Val Lys Ser Asn 675 680 685 Asp Thr LysLys Asn Leu Val Lys Val Thr Gly Ala Leu Tyr Gly Asp 690 695 700 Tyr SerLeu Gly Ser Asn Asp Asn Ala Phe Ile Pro Val Pro Ala Leu 705 710 715 720Thr Pro Ala Ser Leu Ile Ile Glu Asp Asn Asn Tyr Gly Asp Lys Asn 725 730735 Ile Ser Val Leu Ala Pro Thr Asn Asn Asp Met Phe Ile Leu Pro Gly 740745 750 Asn Val Tyr Pro Val Glu Ile Glu Thr Lys Val Ser Val Ser Tyr Ile755 760 765 Gly Arg Gly Phe Asp Lys Asn Gly Thr Pro Leu Ser Gly Ala HisVal 770 775 780 Leu Asn Glu Pro His Val Ile Leu Asp Glu Asp Gly Gly PheSer Phe 785 790 795 800 Glu Tyr Thr Gly Asn Glu Lys Thr Leu Phe Leu LeuLys Gly Arg Thr 805 810 815 Ile Tyr Thr Cys Gln Leu Gly Lys Asn Lys ValHis Lys Gly Ile Val 820 825 830 Phe Val Gly Asp Val Ile Cys Asp Val AsnSer Thr Ser Ser Leu Pro 835 840 845 Asp Glu Phe Val Lys Asn Pro Arg ValGln Asp Leu Leu Ala Lys Asn 850 855 860 Asp Lys Gly 865 <210> SEQ ID NO7 <211> LENGTH: 330 <212> TYPE: DNA <213> ORGANISM: E. coli <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(330) <400> SEQUENCE:7 atc agt aag ttg gca gca tca cct gta ttt ctt gaa aga ggg gtg aat 48 IleSer Lys Leu Ala Ala Ser Pro Val Phe Leu Glu Arg Gly Val Asn 1 5 10 15ata tct gta aga ata cag aag caa att tta tca gaa aaa cca tat gtt 96 IleSer Val Arg Ile Gln Lys Gln Ile Leu Ser Glu Lys Pro Tyr Val 20 25 30 gcattc aga ttg aac gga gac ata cta aga cat tta aag gat gca ttg 144 Ala PheArg Leu Asn Gly Asp Ile Leu Arg His Leu Lys Asp Ala Leu 35 40 45 atg ataata tat ggt atg tca aaa ata gat acc aat gat tgt aga aat 192 Met Ile IleTyr Gly Met Ser Lys Ile Asp Thr Asn Asp Cys Arg Asn 50 55 60 atg tca aggaaa ata atg aaa aca gaa gtg gat aaa acc tta ctg gat 240 Met Ser Arg LysIle Met Lys Thr Glu Val Asp Lys Thr Leu Leu Asp 65 70 75 80 gta tta aaaaat ata aat agc tat gat gac tca gct ttt ata tct aat 288 Val Leu Lys AsnIle Asn Ser Tyr Asp Asp Ser Ala Phe Ile Ser Asn 85 90 95 ttg ata tat ttaatt tca aag atc gag aat aat aaa aaa taa 330 Leu Ile Tyr Leu Ile Ser LysIle Glu Asn Asn Lys Lys * 100 105 <210> SEQ ID NO 8 <211> LENGTH: 109<212> TYPE: PRT <213> ORGANISM: E. coli <400> SEQUENCE: 8 Ile Ser LysLeu Ala Ala Ser Pro Val Phe Leu Glu Arg Gly Val Asn 1 5 10 15 Ile SerVal Arg Ile Gln Lys Gln Ile Leu Ser Glu Lys Pro Tyr Val 20 25 30 Ala PheArg Leu Asn Gly Asp Ile Leu Arg His Leu Lys Asp Ala Leu 35 40 45 Met IleIle Tyr Gly Met Ser Lys Ile Asp Thr Asn Asp Cys Arg Asn 50 55 60 Met SerArg Lys Ile Met Lys Thr Glu Val Asp Lys Thr Leu Leu Asp 65 70 75 80 ValLeu Lys Asn Ile Asn Ser Tyr Asp Asp Ser Ala Phe Ile Ser Asn 85 90 95 LeuIle Tyr Leu Ile Ser Lys Ile Glu Asn Asn Lys Lys 100 105 <210> SEQ ID NO9 <211> LENGTH: 1086 <212> TYPE: DNA <213> ORGANISM: E. coli <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1086) <400>SEQUENCE: 9 atg aat aag att tta ttt att ttt aca ttg ttt ttc tct tca gtactt 48 Met Asn Lys Ile Leu Phe Ile Phe Thr Leu Phe Phe Ser Ser Val Leu 15 10 15 ttt aca ttt gct gta tcg gca gat aaa att ccc gga gat gaa agc ata96 Phe Thr Phe Ala Val Ser Ala Asp Lys Ile Pro Gly Asp Glu Ser Ile 20 2530 act aat att ttt ggc ccg cgt gac agg aac gaa tct tcc ccc aaa cat 144Thr Asn Ile Phe Gly Pro Arg Asp Arg Asn Glu Ser Ser Pro Lys His 35 40 45aat ata tta aat aac cat att aca gca tac agt gaa agt cat act ctg 192 AsnIle Leu Asn Asn His Ile Thr Ala Tyr Ser Glu Ser His Thr Leu 50 55 60 tatgat agg atg act ttt tta tgt ttg tct tct cac aat aca ctt aat 240 Tyr AspArg Met Thr Phe Leu Cys Leu Ser Ser His Asn Thr Leu Asn 65 70 75 80 ggagca tgt cca acc agt gag aat cct agc agt tca tcg gtc agc ggt 288 Gly AlaCys Pro Thr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser Gly 85 90 95 gaa acaaat ata aca tta caa ttt acg gaa aaa aga agt tta ata aaa 336 Glu Thr AsnIle Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys 100 105 110 aga gagcta caa att aaa ggc tat aaa caa tta ttg ttc aaa agt gtt 384 Arg Glu LeuGln Ile Lys Gly Tyr Lys Gln Leu Leu Phe Lys Ser Val 115 120 125 aac tgccca tcc ggc cta aca ctt aac tca gct cat ttt aac tgt aat 432 Asn Cys ProSer Gly Leu Thr Leu Asn Ser Ala His Phe Asn Cys Asn 130 135 140 aaa aacgcg gct tca ggt gca agt tta tat tta tat att cct gct ggc 480 Lys Asn AlaAla Ser Gly Ala Ser Leu Tyr Leu Tyr Ile Pro Ala Gly 145 150 155 160 gaacta aaa aat ttg cct ttt ggt ggt atc tgg gat gct act ctg aag 528 Glu LeuLys Asn Leu Pro Phe Gly Gly Ile Trp Asp Ala Thr Leu Lys 165 170 175 ttaaga gta aaa aga cga tat agt gag acc tat gga act tac act ata 576 Leu ArgVal Lys Arg Arg Tyr Ser Glu Thr Tyr Gly Thr Tyr Thr Ile 180 185 190 aatatc act att aaa tta act gat aag gga aat att cag ata tgg tta 624 Asn IleThr Ile Lys Leu Thr Asp Lys Gly Asn Ile Gln Ile Trp Leu 195 200 205 cctcag ttc aaa agt gac gct cgc gtc gat ctt aac ttg cgt cca act 672 Pro GlnPhe Lys Ser Asp Ala Arg Val Asp Leu Asn Leu Arg Pro Thr 210 215 220 ggtggg ggc aca tat att gga aga aat tct gtt gat atg tgc ttt tat 720 Gly GlyGly Thr Tyr Ile Gly Arg Asn Ser Val Asp Met Cys Phe Tyr 225 230 235 240gat gga tat agt act aac agc agc tct ttg gag ata aga ttt cag gat 768 AspGly Tyr Ser Thr Asn Ser Ser Ser Leu Glu Ile Arg Phe Gln Asp 245 250 255aac aat cct aaa tct gat ggg aaa ttt tat cta agg aaa ata aat gat 816 AsnAsn Pro Lys Ser Asp Gly Lys Phe Tyr Leu Arg Lys Ile Asn Asp 260 265 270gac acc aaa gaa att gca tat act ttg tca ctt ctc ttg gcg ggt aaa 864 AspThr Lys Glu Ile Ala Tyr Thr Leu Ser Leu Leu Leu Ala Gly Lys 275 280 285agt tta act cca aca aat gga acg tca tta aat att gct gac gca gct 912 SerLeu Thr Pro Thr Asn Gly Thr Ser Leu Asn Ile Ala Asp Ala Ala 290 295 300tct ctg gaa aca aac tgg aat aga att aca gct gtc acc atg cca gaa 960 SerLeu Glu Thr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu 305 310 315320 atc agt gtt ccg gtg ttg tgt tgg cct gga cgt ttg caa ttg gat gca 1008Ile Ser Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala 325 330335 aaa gtg gaa aat ccc gag gct gga caa tat atg ggt aat att aat gtt 1056Lys Val Glu Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile Asn Val 340 345350 act ttc aca cca agt agt caa aca ctc tag 1086 Thr Phe Thr Pro Ser SerGln Thr Leu * 355 360 <210> SEQ ID NO 10 <211> LENGTH: 361 <212> TYPE:PRT <213> ORGANISM: E. coli <400> SEQUENCE: 10 Met Asn Lys Ile Leu PheIle Phe Thr Leu Phe Phe Ser Ser Val Leu 1 5 10 15 Phe Thr Phe Ala ValSer Ala Asp Lys Ile Pro Gly Asp Glu Ser Ile 20 25 30 Thr Asn Ile Phe GlyPro Arg Asp Arg Asn Glu Ser Ser Pro Lys His 35 40 45 Asn Ile Leu Asn AsnHis Ile Thr Ala Tyr Ser Glu Ser His Thr Leu 50 55 60 Tyr Asp Arg Met ThrPhe Leu Cys Leu Ser Ser His Asn Thr Leu Asn 65 70 75 80 Gly Ala Cys ProThr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser Gly 85 90 95 Glu Thr Asn IleThr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys 100 105 110 Arg Glu LeuGln Ile Lys Gly Tyr Lys Gln Leu Leu Phe Lys Ser Val 115 120 125 Asn CysPro Ser Gly Leu Thr Leu Asn Ser Ala His Phe Asn Cys Asn 130 135 140 LysAsn Ala Ala Ser Gly Ala Ser Leu Tyr Leu Tyr Ile Pro Ala Gly 145 150 155160 Glu Leu Lys Asn Leu Pro Phe Gly Gly Ile Trp Asp Ala Thr Leu Lys 165170 175 Leu Arg Val Lys Arg Arg Tyr Ser Glu Thr Tyr Gly Thr Tyr Thr Ile180 185 190 Asn Ile Thr Ile Lys Leu Thr Asp Lys Gly Asn Ile Gln Ile TrpLeu 195 200 205 Pro Gln Phe Lys Ser Asp Ala Arg Val Asp Leu Asn Leu ArgPro Thr 210 215 220 Gly Gly Gly Thr Tyr Ile Gly Arg Asn Ser Val Asp MetCys Phe Tyr 225 230 235 240 Asp Gly Tyr Ser Thr Asn Ser Ser Ser Leu GluIle Arg Phe Gln Asp 245 250 255 Asn Asn Pro Lys Ser Asp Gly Lys Phe TyrLeu Arg Lys Ile Asn Asp 260 265 270 Asp Thr Lys Glu Ile Ala Tyr Thr LeuSer Leu Leu Leu Ala Gly Lys 275 280 285 Ser Leu Thr Pro Thr Asn Gly ThrSer Leu Asn Ile Ala Asp Ala Ala 290 295 300 Ser Leu Glu Thr Asn Trp AsnArg Ile Thr Ala Val Thr Met Pro Glu 305 310 315 320 Ile Ser Val Pro ValLeu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala 325 330 335 Lys Val Glu AsnPro Glu Ala Gly Gln Tyr Met Gly Asn Ile Asn Val 340 345 350 Thr Phe ThrPro Ser Ser Gln Thr Leu 355 360 <210> SEQ ID NO 11 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 11 gttgacccta caattgatattttgcaagc 29 <210> SEQ ID NO 12 <211> LENGTH: 30 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:PCR Primer <400> SEQUENCE: 12 cgaccccact ataattcccg ccgttggtgc 30 <210>SEQ ID NO 13 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400>SEQUENCE: 13 gtgatatgtt ttgttcactt ggtaaagatc 30 <210> SEQ ID NO 14<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 14ctcatggctc catttgttgc aaatgcaaac tttatg 36 <210> SEQ ID NO 15 <211>LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 15gggatcgatc ccggggcggc cgcgggcccg gtaccaggcc ttctagaaag cttgacgtcg 60<210> SEQ ID NO 16 <211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 16 cccgctagcg gcgcgcctcg cgaggatccg tcgacgacgtcaagctttct agaaggcctg 60 g 61 <210> SEQ ID NO 17 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 17 aagcttgacg tcgtcgacgg 20<210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 18 cccgctagcg gcgcgcctcg cg 22 <210> SEQ ID NO 19 <211>LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 19ccgtgctgac tctacacccc cagatg 26 <210> SEQ ID NO 20 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 20 gcacatagag aggatagtaacgccg 25 <210> SEQ ID NO 21 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:PCR Primer <400> SEQUENCE: 21 cggtcattgt tggccgtgcg ctgcc 25 <210> SEQID NO 22 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400>SEQUENCE: 22 cacgcagcgc gctgatgcct tccacgcg 28 <210> SEQ ID NO 23 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 23catatttgat atctgagata tctgg 25 <210> SEQ ID NO 24 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 24 tgttgcattc agattgaacg gag 23<210> SEQ ID NO 25 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 25 tattatgatt cataaataca ctgt 24 <210> SEQ ID NO 26<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 26tgtgggtatt tgtttggaca tcgcagcatt aaatataaaa atagcacagg 50 <210> SEQ IDNO 27 <211> LENGTH: 7239 <212> TYPE: DNA <213> ORGANISM: E. coli <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (283)...(999) <221>NAME/KEY: CDS <222> LOCATION: (1028)...(1531) <221> NAME/KEY: CDS <222>LOCATION: (1589)...(4192) <221> NAME/KEY: CDS <222> LOCATION:(4196)...(5281) <221> NAME/KEY: CDS <222> LOCATION: (5790)...(6119)<400> SEQUENCE: 27 atatatctta ttgaggaata tcggtgtcat tgagtaccgttaacttaaga taaagaatct 60 gtctggaaat cgcaggacca agaactctca gtacatctgtggcgataata ttatcgcttc 120 ttatacattc caatatgcag ttcttgtggg tatttgtttggacatcgcag cattaaatat 180 aaaaatagca caggaggcat aattatttgt ttttactgtcttattttttt atcccatttt 240 tttttgtttt gatttatctt tgatgaaagc tcaggagggaatatg cat aaa tta ttt 297 His Lys Leu Phe 1 tgt tta cta agt tta ctc ataact cca ttt gtt gca aat gca aac ttt 345 Cys Leu Leu Ser Leu Leu Ile ThrPro Phe Val Ala Asn Ala Asn Phe 5 10 15 20 atg ata tat cca ata tca aaagat tta aag aat gga aat agc gag tta 393 Met Ile Tyr Pro Ile Ser Lys AspLeu Lys Asn Gly Asn Ser Glu Leu 25 30 35 att cgt gtt tat tca aaa tca aaagag ata caa tat ata aaa ata tat 441 Ile Arg Val Tyr Ser Lys Ser Lys GluIle Gln Tyr Ile Lys Ile Tyr 40 45 50 aca aaa aag att att aat ccc ggc acaact gaa gaa cat gaa gtt gat 489 Thr Lys Lys Ile Ile Asn Pro Gly Thr ThrGlu Glu His Glu Val Asp 55 60 65 atg ccc aat tgg gat ggt ggg ttt gta gttact cct caa aaa gtt att 537 Met Pro Asn Trp Asp Gly Gly Phe Val Val ThrPro Gln Lys Val Ile 70 75 80 ctt cct gca gga ggg agt aaa tca ata cgt ttaact caa ttt aga ata 585 Leu Pro Ala Gly Gly Ser Lys Ser Ile Arg Leu ThrGln Phe Arg Ile 85 90 95 100 cca aaa aaa gag gaa att tat aga gta tat tttgag gcg gta aaa cca 633 Pro Lys Lys Glu Glu Ile Tyr Arg Val Tyr Phe GluAla Val Lys Pro 105 110 115 gat agc aaa gaa aat gta att gat aat aaa aaacta aca aca gag cta 681 Asp Ser Lys Glu Asn Val Ile Asp Asn Lys Lys LeuThr Thr Glu Leu 120 125 130 tct gtt aat ata att tat gcg gct cta atc agatct tta cca agt gaa 729 Ser Val Asn Ile Ile Tyr Ala Ala Leu Ile Arg SerLeu Pro Ser Glu 135 140 145 caa aac ata tca cta aac att tct aga aat gcaaga aaa aat ata att 777 Gln Asn Ile Ser Leu Asn Ile Ser Arg Asn Ala ArgLys Asn Ile Ile 150 155 160 att tat aat aat ggg aat gtt aga gca ggt gttaaa gat att tat ttt 825 Ile Tyr Asn Asn Gly Asn Val Arg Ala Gly Val LysAsp Ile Tyr Phe 165 170 175 180 tgt aag tca tct aat atc gat gat agc tgtgta aaa aaa acg cat aac 873 Cys Lys Ser Ser Asn Ile Asp Asp Ser Cys ValLys Lys Thr His Asn 185 190 195 aag aat ata tat cca gaa aag tca ttt gatacg ctg gtt aat aac aat 921 Lys Asn Ile Tyr Pro Glu Lys Ser Phe Asp ThrLeu Val Asn Asn Asn 200 205 210 ttt tct tat gtt ttc att aaa tta aac catgaa gac ata gaa aaa gag 969 Phe Ser Tyr Val Phe Ile Lys Leu Asn His GluAsp Ile Glu Lys Glu 215 220 225 caa gga cta ata caa tta aaa gtt cct tgatta ctcatctata tactaaggag 1022 Gln Gly Leu Ile Gln Leu Lys Val Pro * Leu230 235 ttctaatgaa attaaaaaaa actattggtg caatg gca ctg acc aca atg ttt1075 Ala Leu Thr Thr Met Phe 240 gta gct atg agt gct tct gca gta gag aaaaat atc act gta aca gct 1123 Val Ala Met Ser Ala Ser Ala Val Glu Lys AsnIle Thr Val Thr Ala 245 250 255 260 agt gtt gat cct aca att gat att ttgcaa gct gat ggt agt agt tta 1171 Ser Val Asp Pro Thr Ile Asp Ile Leu GlnAla Asp Gly Ser Ser Leu 265 270 275 cct act gct gta gaa tta acc tat tcacct gcg gca agt cgt ttt gaa 1219 Pro Thr Ala Val Glu Leu Thr Tyr Ser ProAla Ala Ser Arg Phe Glu 280 285 290 aat tat aaa atc gca act aaa gtt cataca aat gtt ata aat aaa aat 1267 Asn Tyr Lys Ile Ala Thr Lys Val His ThrAsn Val Ile Asn Lys Asn 295 300 305 gta cta gtt aag ctt gta aat gat ccaaaa ctt aca aat gtt ttg gat 1315 Val Leu Val Lys Leu Val Asn Asp Pro LysLeu Thr Asn Val Leu Asp 310 315 320 tct aca aaa caa ctc ccc att act gtatca tat gga gga aag act cta 1363 Ser Thr Lys Gln Leu Pro Ile Thr Val SerTyr Gly Gly Lys Thr Leu 325 330 335 340 tca acc gca gat gtg act ttt gaacct gca gaa tta aat ttt gga acg 1411 Ser Thr Ala Asp Val Thr Phe Glu ProAla Glu Leu Asn Phe Gly Thr 345 350 355 tca ggt gta act ggt gta tct tcttcc caa gat tta gtg att ggt gcg 1459 Ser Gly Val Thr Gly Val Ser Ser SerGln Asp Leu Val Ile Gly Ala 360 365 370 act aca gca caa gca cca acg gcggga aat tat agt ggg gtc gtt tct 1507 Thr Thr Ala Gln Ala Pro Thr Ala GlyAsn Tyr Ser Gly Val Val Ser 375 380 385 atc tta atg acc tta gca tca taaata ttttaatata taaaggagca 1554 Ile Leu Met Thr Leu Ala Ser * Ile 390 395ggcacactgc tccttattat atggcaataa taaaatg aca aaa aaa aat aca tta 1609Thr Lys Lys Asn Thr Leu 400 tat ata acg atc atc gca atg cta act cca tattca gtt ttt tcc gga 1657 Tyr Ile Thr Ile Ile Ala Met Leu Thr Pro Tyr SerVal Phe Ser Gly 405 410 415 gat ata ccc aac tct ttc cgt gat tta tgg ggagaa caa gat gaa ttt 1705 Asp Ile Pro Asn Ser Phe Arg Asp Leu Trp Gly GluGln Asp Glu Phe 420 425 430 tat gaa gta aaa cta tat gga caa act cta ggaata cat cga att aaa 1753 Tyr Glu Val Lys Leu Tyr Gly Gln Thr Leu Gly IleHis Arg Ile Lys 435 440 445 450 aca acc cca aca cat att aag ttt tat tcaccc gaa agc att tta gat 1801 Thr Thr Pro Thr His Ile Lys Phe Tyr Ser ProGlu Ser Ile Leu Asp 455 460 465 aaa ata aat gta aaa aaa gaa aag gaa aagaaa ttg agt gtt ttg ttc 1849 Lys Ile Asn Val Lys Lys Glu Lys Glu Lys LysLeu Ser Val Leu Phe 470 475 480 act aat tct ttt tca aga aat ggc aat atgagt tgt cag ggg aat gct 1897 Thr Asn Ser Phe Ser Arg Asn Gly Asn Met SerCys Gln Gly Asn Ala 485 490 495 act ata cag tat aac tgc aat tac att aaaaca aaa tca gta gat gtc 1945 Thr Ile Gln Tyr Asn Cys Asn Tyr Ile Lys ThrLys Ser Val Asp Val 500 505 510 atc gtt gat gat gtt gat aat gtt gtt aacctt ttt ata ggt aat gaa 1993 Ile Val Asp Asp Val Asp Asn Val Val Asn LeuPhe Ile Gly Asn Glu 515 520 525 530 ttt ctg gat tct gaa gca cac aat gatgaa tat cat caa tta tca cga 2041 Phe Leu Asp Ser Glu Ala His Asn Asp GluTyr His Gln Leu Ser Arg 535 540 545 aat gta aaa aaa gct ttt ata caa agccag aca att aat gtc tca gat 2089 Asn Val Lys Lys Ala Phe Ile Gln Ser GlnThr Ile Asn Val Ser Asp 550 555 560 tct ggg aag tat aaa agt ttg tct gtttca ggg aat agc gcg ctg ggt 2137 Ser Gly Lys Tyr Lys Ser Leu Ser Val SerGly Asn Ser Ala Leu Gly 565 570 575 att aca gat aca agt tat gct gtc ttaaat tgg tgg atg aat tac aat 2185 Ile Thr Asp Thr Ser Tyr Ala Val Leu AsnTrp Trp Met Asn Tyr Asn 580 585 590 aaa ttt aat ggt tac agc aac aac gaaaga aca atc aat agt ttg tac 2233 Lys Phe Asn Gly Tyr Ser Asn Asn Glu ArgThr Ile Asn Ser Leu Tyr 595 600 605 610 ttt aga cat gat tta gat aag agatat tat tat caa ttt gga cga atg 2281 Phe Arg His Asp Leu Asp Lys Arg TyrTyr Tyr Gln Phe Gly Arg Met 615 620 625 gat cgt aca gat ttg tca caa agtatt agc ggg aac ttt aat ttt aac 2329 Asp Arg Thr Asp Leu Ser Gln Ser IleSer Gly Asn Phe Asn Phe Asn 630 635 640 tta ctt cct tta ccc gat att gatggt ata agg aca gga acc aca caa 2377 Leu Leu Pro Leu Pro Asp Ile Asp GlyIle Arg Thr Gly Thr Thr Gln 645 650 655 tct tat atc aaa aat aca gat aagttt atc gca tcc cct gta act gtt 2425 Ser Tyr Ile Lys Asn Thr Asp Lys PheIle Ala Ser Pro Val Thr Val 660 665 670 atg tta act aat ttt tcc aga gtggaa gct ttt cgc aat aat caa tta 2473 Met Leu Thr Asn Phe Ser Arg Val GluAla Phe Arg Asn Asn Gln Leu 675 680 685 690 ttg ggc gta tgg tat tta gattct gga gta aat gaa tta gat aca gct 2521 Leu Gly Val Trp Tyr Leu Asp SerGly Val Asn Glu Leu Asp Thr Ala 695 700 705 cgt tta cct tat ggt agt tacgat ctt aaa tta aaa att ttt gaa aat 2569 Arg Leu Pro Tyr Gly Ser Tyr AspLeu Lys Leu Lys Ile Phe Glu Asn 710 715 720 act cag tta gtt cgt gaa gaaata att cct ttt aat aaa ggg aga agt 2617 Thr Gln Leu Val Arg Glu Glu IleIle Pro Phe Asn Lys Gly Arg Ser 725 730 735 tct att ggt gat atg caa tgggac gtt ttc att cag gga ggg aat att 2665 Ser Ile Gly Asp Met Gln Trp AspVal Phe Ile Gln Gly Gly Asn Ile 740 745 750 att aat gac aag gat cgt tacata gaa aaa caa aat aat cat aag tca 2713 Ile Asn Asp Lys Asp Arg Tyr IleGlu Lys Gln Asn Asn His Lys Ser 755 760 765 770 tca gtt aat gct ggg ctacgt tta cca att acg aaa aat atc tct gtt 2761 Ser Val Asn Ala Gly Leu ArgLeu Pro Ile Thr Lys Asn Ile Ser Val 775 780 785 caa caa gga gca tct gttata gat aat aaa aat tat tat gaa ggg agt 2809 Gln Gln Gly Ala Ser Val IleAsp Asn Lys Asn Tyr Tyr Glu Gly Ser 790 795 800 ctc aaa tgg aat tcc ggcatt ctg tct ggc tca cta aat agt gag ttc 2857 Leu Lys Trp Asn Ser Gly IleLeu Ser Gly Ser Leu Asn Ser Glu Phe 805 810 815 agt ttt ctt tgg gga gataat gca aaa ggt aat tat caa agt atc tcg 2905 Ser Phe Leu Trp Gly Asp AsnAla Lys Gly Asn Tyr Gln Ser Ile Ser 820 825 830 tat acc gat gga ttt agttta tca ttt tat cat aat gat aag cgg gtc 2953 Tyr Thr Asp Gly Phe Ser LeuSer Phe Tyr His Asn Asp Lys Arg Val 835 840 845 850 gat aat tgt gga agaaat tac aat gct ggt tgg agt gga tgc tac gaa 3001 Asp Asn Cys Gly Arg AsnTyr Asn Ala Gly Trp Ser Gly Cys Tyr Glu 855 860 865 tca tat tcg gca tcttta agt att cct tta ttg gga tgg aca agt act 3049 Ser Tyr Ser Ala Ser LeuSer Ile Pro Leu Leu Gly Trp Thr Ser Thr 870 875 880 ctg gga tat agt gacact tat agt gaa tca gtt tat aaa aac cat att 3097 Leu Gly Tyr Ser Asp ThrTyr Ser Glu Ser Val Tyr Lys Asn His Ile 885 890 895 ctt tct gaa tat ggtttt tat aat caa aac ata tat aaa ggg aga acc 3145 Leu Ser Glu Tyr Gly PheTyr Asn Gln Asn Ile Tyr Lys Gly Arg Thr 900 905 910 caa aga tgg caa ctgact tcg tcc acc tct tta aaa tgg atg gat tat 3193 Gln Arg Trp Gln Leu ThrSer Ser Thr Ser Leu Lys Trp Met Asp Tyr 915 920 925 930 aat ttt atg ccagca att gga ata tat aac agt gag caa aga caa ctg 3241 Asn Phe Met Pro AlaIle Gly Ile Tyr Asn Ser Glu Gln Arg Gln Leu 935 940 945 act gat aaa ggcgga tat ata tct gta act ctc acc cga gcc agc aga 3289 Thr Asp Lys Gly GlyTyr Ile Ser Val Thr Leu Thr Arg Ala Ser Arg 950 955 960 gaa aat tca ttaaac gca ggg tat tct tac aac tat tcc aga gga aag 3337 Glu Asn Ser Leu AsnAla Gly Tyr Ser Tyr Asn Tyr Ser Arg Gly Lys 965 970 975 tat tct tct aacgaa tta ttt gtt gat gga tat atg aca tca aca aat 3385 Tyr Ser Ser Asn GluLeu Phe Val Asp Gly Tyr Met Thr Ser Thr Asn 980 985 990 aat ggt gac tatcat gag gta aga atg cgt ttt aat aaa aat aga cat 3433 Asn Gly Asp Tyr HisGlu Val Arg Met Arg Phe Asn Lys Asn Arg His 995 1000 1005 1010 aat gcagaa ggt aga ctt tca ggt cgt ata aac aat cga ttt gga gat 3481 Asn Ala GluGly Arg Leu Ser Gly Arg Ile Asn Asn Arg Phe Gly Asp 1015 1020 1025 ttaaat ggt tca ttc agc atg aat aaa aac aga aac acc aac agt agc 3529 Leu AsnGly Ser Phe Ser Met Asn Lys Asn Arg Asn Thr Asn Ser Ser 1030 1035 1040aat cat tct ctc act ggt ggt tat aat tcc tca ttt gct ctt aca agt 3577 AsnHis Ser Leu Thr Gly Gly Tyr Asn Ser Ser Phe Ala Leu Thr Ser 1045 10501055 gat gga ttt tac tgg gga gga agt gca tct ggt ttg aca aaa cta gct3625 Asp Gly Phe Tyr Trp Gly Gly Ser Ala Ser Gly Leu Thr Lys Leu Ala1060 1065 1070 ggc ggt att atc aag gtt aaa tca aac gat act aaa aaa aatctg gta 3673 Gly Gly Ile Ile Lys Val Lys Ser Asn Asp Thr Lys Lys Asn LeuVal 1075 1080 1085 1090 aaa gtg act ggg gca ttg tac ggt gat tat tcg ctaggg agc aac gat 3721 Lys Val Thr Gly Ala Leu Tyr Gly Asp Tyr Ser Leu GlySer Asn Asp 1095 1100 1105 aat gct ttt att cct gta cca gca tta act ccagcc agt tta att att 3769 Asn Ala Phe Ile Pro Val Pro Ala Leu Thr Pro AlaSer Leu Ile Ile 1110 1115 1120 gaa gat aat aat tat ggt gac aag aat atttct gta ctt gca cca acg 3817 Glu Asp Asn Asn Tyr Gly Asp Lys Asn Ile SerVal Leu Ala Pro Thr 1125 1130 1135 aac aac gat atg ttt ata ttg ccg ggtaat gtt tat cct gtt gaa att 3865 Asn Asn Asp Met Phe Ile Leu Pro Gly AsnVal Tyr Pro Val Glu Ile 1140 1145 1150 gaa acc aaa gta agt gtt tct tatatt ggt aga ggt ttt gac aaa aac 3913 Glu Thr Lys Val Ser Val Ser Tyr IleGly Arg Gly Phe Asp Lys Asn 1155 1160 1165 1170 ggc acg cca ctt tct ggcgca cat gtt ttg aat gaa cca cat gtt atc 3961 Gly Thr Pro Leu Ser Gly AlaHis Val Leu Asn Glu Pro His Val Ile 1175 1180 1185 ctg gat gag gac ggtgga ttt tcg ttt gaa tat aca ggt aat gag aaa 4009 Leu Asp Glu Asp Gly GlyPhe Ser Phe Glu Tyr Thr Gly Asn Glu Lys 1190 1195 1200 aca ctt ttt ttatta aag ggc aga act att tat aca tgt caa ctg ggg 4057 Thr Leu Phe Leu LeuLys Gly Arg Thr Ile Tyr Thr Cys Gln Leu Gly 1205 1210 1215 aaa aat aaagtt cac aaa ggc att gtt ttc gtc gga gat gtt ata tgt 4105 Lys Asn Lys ValHis Lys Gly Ile Val Phe Val Gly Asp Val Ile Cys 1220 1225 1230 gat gttaat agc aca agt tcc tta cca gat gaa ttt gta aag aac cca 4153 Asp Val AsnSer Thr Ser Ser Leu Pro Asp Glu Phe Val Lys Asn Pro 1235 1240 1245 1250cgt gtg cag gat ttg ctg gca aag aat gat aaa gga taa acg 4195 Arg Val GlnAsp Leu Leu Ala Lys Asn Asp Lys Gly * Thr 1255 1260 atg aat aag att ttattt att ttt aca ttg ttt ttc tct tca gta ctt 4243 Asn Lys Ile Leu Phe IlePhe Thr Leu Phe Phe Ser Ser Val Leu 1265 1270 1275 ttt aca ttt gct gtatcg gca gat aaa att ccc gga gat gaa agc ata 4291 Phe Thr Phe Ala Val SerAla Asp Lys Ile Pro Gly Asp Glu Ser Ile 1280 1285 1290 act aat att tttggc ccg cgt gac agg aac gaa tct tcc ccc aaa cat 4339 Thr Asn Ile Phe GlyPro Arg Asp Arg Asn Glu Ser Ser Pro Lys His 1295 1300 1305 1310 aat atatta aat aac cat att aca gca tac agt gaa agt cat act ctg 4387 Asn Ile LeuAsn Asn His Ile Thr Ala Tyr Ser Glu Ser His Thr Leu 1315 1320 1325 tatgat agg atg act ttt tta tgt ttg tct tct cac aat aca ctt aat 4435 Tyr AspArg Met Thr Phe Leu Cys Leu Ser Ser His Asn Thr Leu Asn 1330 1335 1340gga gca tgt cca acc agt gag aat cct agc agt tca tcg gtc agc ggt 4483 GlyAla Cys Pro Thr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser Gly 1345 13501355 gaa aca aat ata aca tta caa ttt acg gaa aaa aga agt tta ata aaa4531 Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys1360 1365 1370 aga gag cta caa att aaa ggc tat aaa caa tta ttg ttc aaaagt gtt 4579 Arg Glu Leu Gln Ile Lys Gly Tyr Lys Gln Leu Leu Phe Lys SerVal 1375 1380 1385 1390 aac tgc cca tcc ggc cta aca ctt aac tca gct catttt aac tgt aat 4627 Asn Cys Pro Ser Gly Leu Thr Leu Asn Ser Ala His PheAsn Cys Asn 1395 1400 1405 aaa aac gcg gct tca ggt gca agt tta tat ttatat att cct gct ggc 4675 Lys Asn Ala Ala Ser Gly Ala Ser Leu Tyr Leu TyrIle Pro Ala Gly 1410 1415 1420 gaa cta aaa aat ttg cct ttt ggt ggt atctgg gat gct act ctg aag 4723 Glu Leu Lys Asn Leu Pro Phe Gly Gly Ile TrpAsp Ala Thr Leu Lys 1425 1430 1435 tta aga gta aaa aga cga tat agt gagacc tat gga act tac act ata 4771 Leu Arg Val Lys Arg Arg Tyr Ser Glu ThrTyr Gly Thr Tyr Thr Ile 1440 1445 1450 aat atc act att aaa tta act gataag gga aat att cag ata tgg tta 4819 Asn Ile Thr Ile Lys Leu Thr Asp LysGly Asn Ile Gln Ile Trp Leu 1455 1460 1465 1470 cct cag ttc aaa agt gacgct cgc gtc gat ctt aac ttg cgt cca act 4867 Pro Gln Phe Lys Ser Asp AlaArg Val Asp Leu Asn Leu Arg Pro Thr 1475 1480 1485 ggt ggg ggc aca tatatt gga aga aat tct gtt gat atg tgc ttt tat 4915 Gly Gly Gly Thr Tyr IleGly Arg Asn Ser Val Asp Met Cys Phe Tyr 1490 1495 1500 gat gga tat agtact aac agc agc tct ttg gag ata aga ttt cag gat 4963 Asp Gly Tyr Ser ThrAsn Ser Ser Ser Leu Glu Ile Arg Phe Gln Asp 1505 1510 1515 aac aat cctaaa tct gat ggg aaa ttt tat cta agg aaa ata aat gat 5011 Asn Asn Pro LysSer Asp Gly Lys Phe Tyr Leu Arg Lys Ile Asn Asp 1520 1525 1530 gac accaaa gaa att gca tat act ttg tca ctt ctc ttg gcg ggt aaa 5059 Asp Thr LysGlu Ile Ala Tyr Thr Leu Ser Leu Leu Leu Ala Gly Lys 1535 1540 1545 1550agt tta act cca aca aat gga acg tca tta aat att gct gac gca gct 5107 SerLeu Thr Pro Thr Asn Gly Thr Ser Leu Asn Ile Ala Asp Ala Ala 1555 15601565 tct ctg gaa aca aac tgg aat aga att aca gct gtc acc atg cca gaa5155 Ser Leu Glu Thr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu1570 1575 1580 atc agt gtt ccg gtg ttg tgt tgg cct gga cgt ttg caa ttggat gca 5203 Ile Ser Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu AspAla 1585 1590 1595 aaa gtg gaa aat ccc gag gct gga caa tat atg ggt aatatt aat gtt 5251 Lys Val Glu Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn IleAsn Val 1600 1605 1610 act ttc aca cca agt agt caa aca ctc tag ataacaacaatat tggcgctatt 5304 Thr Phe Thr Pro Ser Ser Gln Thr Leu * Ile1615 1620 gcgcgccaat attgtaaagg ggtaatctgt ttgttaacaa aacattttgtttcaattcag 5364 tttgcatcgc aataaatctc tactagagac atttttatac agcatagtattatacaacac 5424 attcaaaata aggatatttt tatccacctt taaaataagt aaaaaactgctttggtataa 5484 caccataatg tttattaaaa accctaataa aataagatgt actggaaattccaatcatat 5544 ttgatatctg agatatctgg tatgaatttt caagtagtaa taacgctgccttgctcattc 5604 tcaattgcat taagaactgg ttaaaattag tattctcaga ttctagtctttttctgatgg 5664 ttatttctga ttcattaaac atatctgcaa tgatagccag tgtccattttctggatagat 5724 ctttttcgat aatatttctg accttgtcag aaaaaaattc acagatgatatataaattga 5784 ttctatta ttt ttt att att ctc gat ctt tga aat taa ata tatcaa att 5834 Phe Phe Ile Ile Leu Asp Leu * Asn * Ile Tyr Gln Ile 16251630 1635 aga tat aaa agc tga gtc atc ata gct att tat att ttt taa tacatc 5882 Arg Tyr Lys Ser * Val Ile Ile Ala Ile Tyr Ile Phe * Tyr Ile1640 1645 1650 cag taa ggt ttt atc cac ttc tgt ttt cat tat ttt cct tgacat att 5930 Gln * Gly Phe Ile His Phe Cys Phe His Tyr Phe Pro * His Ile1655 1660 tct aca atc att ggt atc tat ttt tga cat acc ata tat tat catcaa 5978 Ser Thr Ile Ile Gly Ile Tyr Phe * His Thr Ile Tyr Tyr His Gln1665 1670 1675 tgc atc ctt taa atg tct tag tat gtc tcc gtt caa tct gaatgc aac 6026 Cys Ile Leu * Met Ser * Tyr Val Ser Val Gln Ser Glu Cys Asn1680 1685 1690 ata tgg ttt ttc tga taa aat ttg ctt ctg tat tct tac agatat att 6074 Ile Trp Phe Phe * * Asn Leu Leu Leu Tyr Ser Tyr Arg Tyr Ile1695 1700 1705 cac ccc tct ttc aag aaa tac agg t gatgctgcca acttactgat6119 His Pro Ser Phe Lys Lys Tyr Arg 1710 1715 ttagtgtatg atggtgtttttgaggtgctc cagtggcttc tgtttctatc agctgtccct 6179 cctgttcagc tactgacggggtggtgcgta acggcaaaag cactgccgga catcagcgct 6239 atctctgctc tcactgccgtaaaacatggc aactgcagtt cacttacact gcttctcaac 6299 ccggtacgca ccagaaaatcattgatatgg ccatgaatgg cgttggatgc cgggcaacag 6359 cccgcattat gggcgttggcctcaacacga ttttacgtca cttaaaaaac tcaggccgca 6419 gtcggtaacc tcgcgcatacagccgggcag tgacgtcatc gtctgcgcgg aaatggacga 6479 acagtggggc tatgtcggggctaaatcgcg ccagcgctgg ctgttttacg cgtatgacag 6539 gctccggaag acggttgttgcgcacgtatt cggtgaacgc actatggcga cgctggggcg 6599 tcttatgagc ctgctgtcaccctttgacgt ggtgatatgg atgacggatg gctggccgct 6659 gtatgaatcc cgcctgaagggaaagctgca cgtaatcagc aagcgatata cgcagcgaat 6719 tgagcggcat aacctgaatctgaggcagca cctggcacgg ctgggacgga agtcgctgtc 6779 gttctcaaaa tcggtggagctgcatgacaa agtcatcggg cattatctga acataaaaca 6839 ctatcaataa gttagagtcattacctggtt cacgtattat tatccgtgac tctttcctgg 6899 taactcccgc ataataacctcacttttcca gtattccaga agatgatgtt ttttcctcga 6959 taataaaaat gtgccaatatggaaataaga aatcggattt tttatcagca tacgcaaatt 7019 ttcagataac aatgaatacagatgtatttt atatacacag ataaaaccgc gcaacagaca 7079 taaatatgac agtagcatgaaaaagcagag agagacaggg tgatacagaa aagtaactat 7139 ttttttagct atagtattattggttttacc tattttcgtg attgtgtttc tgtatatttg 7199 acaatgagtc tctcagaatcggtttctcga agtgacgagc 7239 <210> SEQ ID NO 28 <211> LENGTH: 361 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: ETEC Protein Homology Sequence <400> SEQUENCE: 28 Thr MetAsn Lys Ile Leu Phe Ile Phe Thr Leu Phe Phe Ser Ser Val 1 5 10 15 LeuPhe Thr Phe Ala Val Ser Ala Asp Lys Ile Pro Gly Asp Glu Ser 20 25 30 IleThr Asn Ile Phe Gly Pro Arg Asp Arg Asn Glu Ser Ser Pro Lys 35 40 45 HisAsn Ile Leu Asn Asn His Ile Thr Ala Tyr Ser Glu Ser His Thr 50 55 60 LeuTyr Asp Arg Met Thr Phe Leu Cys Leu Ser Ser His Asn Thr Leu 65 70 75 80Asn Gly Ala Cys Pro Thr Ser Glu Asn Pro Ser Ser Ser Ser Val Ser 85 90 95Gly Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile 100 105110 Lys Arg Glu Leu Gln Ile Lys Gly Tyr Lys Gln Leu Leu Phe Lys Ser 115120 125 Val Asn Cys Pro Ser Gly Leu Thr Leu Asn Ser Ala His Phe Asn Cys130 135 140 Asn Lys Asn Ala Ala Ser Gly Ala Ser Leu Tyr Leu Tyr Ile ProAla 145 150 155 160 Gly Glu Leu Lys Asn Leu Pro Phe Gly Gly Ile Trp AspAla Thr Leu 165 170 175 Lys Leu Arg Val Lys Arg Arg Tyr Ser Glu Thr TyrGly Thr Tyr Thr 180 185 190 Ile Asn Ile Thr Ile Lys Leu Thr Asp Lys GlyAsn Ile Gln Ile Trp 195 200 205 Leu Pro Gln Phe Lys Ser Asp Ala Arg ValAsp Leu Asn Leu Arg Pro 210 215 220 Thr Gly Gly Gly Thr Tyr Ile Gly ArgAsn Ser Val Asp Met Cys Phe 225 230 235 240 Tyr Asp Gly Tyr Ser Thr AsnSer Ser Ser Leu Glu Ile Arg Phe Gln 245 250 255 Asp Asn Asn Pro Lys SerAsp Gly Lys Phe Tyr Leu Arg Lys Ile Asn 260 265 270 Asp Asp Thr Lys GluIle Ala Tyr Thr Leu Ser Leu Leu Leu Ala Gly 275 280 285 Ser Leu Thr ProThr Asn Gly Thr Ser Leu Asn Ile Ala Asp Ala Ala 290 295 300 Ser Leu PheThr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu 305 310 315 320 IleSer Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp Ala 325 330 335Lys Val Glu Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile Asn Val 340 345350 Thr Phe Thr Pro Ser Ser Gln Thr Leu 355 360 <210> SEQ ID NO 29 <211>LENGTH: 359 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: ETEC Protein Homology Sequence <400>SEQUENCE: 29 Met Asn Lys Ile Leu Phe Ile Phe Thr Leu Phe Phe Ser Ser GlyPhe 1 5 10 15 Phe Thr Phe Ala Val Ser Ala Asp Lys Asn Pro Gly Ser GluAsn Met 20 25 30 Thr Asn Thr Ile Gly Pro His Asp Arg Gly Gly Ser Ser ProIle Tyr 35 40 45 Asn Ile Leu Asn Ser Tyr Leu Thr Ala Tyr Asn Gly Ser HisHis Leu 50 55 60 Tyr Asp Arg Met Ser Phe Leu Cys Leu Ser Ser Gln Asn ThrLeu Asn 65 70 75 80 Gly Ala Cys Pro Ser Ser Asp Ala Pro Gly Thr Ala ThrIle Asp Gly 85 90 95 Glu Thr Asn Ile Thr Leu Gln Phe Thr Glu Lys Arg SerLeu Ile Lys 100 105 110 Arg Glu Leu Gln Ile Lys Gly Tyr Lys Gln Phe LeuPhe Lys Asn Ala 115 120 125 Asn Cys Pro Ser Lys Leu Ala Leu Asn Ser SerHis Phe Gln Cys Asn 130 135 140 Arg Glu Gln Ala Ser Gly Ala Thr Leu SerLeu Tyr Ile Pro Ala Gly 145 150 155 160 Glu Leu Asn Lys Leu Pro Phe GlyGly Val Trp Asn Ala Val Leu Lys 165 170 175 Leu Asn Val Lys Arg Arg TyrThr Thr Tyr Gly Thr Tyr Thr Ile Asn 180 185 190 Ile Thr Val Asn Leu ThrAsp Lys Gly Asn Ile Gln Ile Trp Leu Pro 195 200 205 Gln Phe Lys Ser AsnAla Arg Val Asp Leu Asn Leu Arg Pro Thr Gly 210 215 220 Gly Gly Thr TyrIle Gly Arg Asn Ser Val Asp Met Cys Phe Tyr Asp 225 230 235 240 Gly TyrSer Thr Met Ser Ser Ser Leu Glu Ile Arg Phe Gln Asp Asp 245 250 255 AsnSer Lys Ser Asp Gly Lys Phe Tyr Leu Lys Lys Ile Asn Asp Asp 260 265 270Ser Lys Glu Leu Val Tyr Thr Leu Ser Leu Leu Leu Ala Gly Lys Asn 275 280285 Leu Thr Pro Thr Asn Gly Gln Ala Leu Asn Ile Asn Thr Ala Ser Leu 290295 300 Glu Thr Asn Trp Asn Arg Ile Thr Ala Val Thr Met Pro Glu Ile Ser305 310 315 320 Val Pro Val Leu Cys Trp Pro Gly Arg Leu Gln Leu Asp AlaLys Val 325 330 335 Lys Asn Pro Glu Ala Gly Gln Tyr Met Gly Asn Ile LysIle Thr Phe 340 345 350 Thr Pro Ser Ser Gln Thr Leu 355 <210> SEQ ID NO30 <211> LENGTH: 364 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: ETEC Protein Homology Sequence<400> SEQUENCE: 30 Met Lys Lys Val Ile Phe Val Leu Ser Met Phe Leu CysSer Gln Val 1 5 10 15 Tyr Gly Gln Ser Trp His Thr Asn Val Glu Ala GlySer Ile Asn Lys 20 25 30 Thr Phe Ser Ile Gly Pro Ile Asp Arg Ser Ala AlaAla Ser Tyr Pro 35 40 45 Ala His Tyr Ile Phe His Glu Asx Val Ala Gly TyrAsn Lys Asp His 50 55 60 Ser Leu Phe Asp Arg Met Thr Phe Leu Cys Met SerSer Thr Asp Ala 65 70 75 80 Ser Lys Gly Ala Cys Pro Thr Gly Glu Asn SerLys Ser Ser Gln Gly 85 90 95 Glu Thr Asn Ile Lys Leu Ile Phe Thr Glu LysLys Ser Leu Ala Arg 100 105 110 Lys Thr Leu Asn Leu Lys Gly Tyr Lys ArgPhe Leu Tyr Glu Ser Asp 115 120 125 Arg Cys Ile His Tyr Val Asp Lys MetAsn Leu Asn Ser His Thr Val 130 135 140 Lys Cys Val Gly Ser Phe Thr ArgGly Val Asp Phe Thr Leu Tyr Ile 145 150 155 160 Pro Gln Gly Glu Ile AspGly Leu Leu Thr Gly Gly Ile Trp Lys Ala 165 170 175 Thr Leu Glu Leu ArgVal Lys Arg His Tyr Asp Tyr Asn His Gly Thr 180 185 190 Tyr Lys Val AsnIle Thr Val Asp Leu Thr Asp Lys Gly Asn Ile Gln 195 200 205 Val Trp ThrPro Lys Phe His Ser Asp Pro Arg Ile Asp Leu Asn Leu 210 215 220 Arg ProGlu Gly Asn Gly Lys Tyr Ser Gly Ser Asn Val Leu Glu Met 225 230 235 240Cys Leu Tyr Asp Gly Tyr Ser Thr His Ser Gln Ser Ile Glu Met Arg 245 250255 Phe Gln Asp Asp Ser Gln Thr Gly Asn Asn Glu Tyr Asn Leu Ile Lys 260265 270 Thr Gly Glu Pro Leu Lys Lys Leu Pro Tyr Lys Leu Ser Leu Leu Leu275 280 285 Gly Gly Arg Glu Phe Tyr Pro Asn Asn Gly Lys Ala Phe Thr IleAsn 290 295 300 Asp Thr Ser Ser Leu Phe Ile Asn Trp Asn Arg Ile Lys SerVal Ser 305 310 315 320 Leu Pro Gln Ile Ser Ile Pro Val Leu Cys Trp ProAla Asn Leu Thr 325 330 335 Phe Met Ser Glu Leu Asn Asn Pro Glu Ala GlyGlu Tyr Ser Gly Ile 340 345 350 Leu Asn Val Thr Phe Thr Pro Ser Ser SerSer Leu 355 360 <210> SEQ ID NO 31 <211> LENGTH: 362 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: ETEC Protein Homology Sequence <400> SEQUENCE: 31 Met LysLys Ile Phe Ile Phe Leu Ser Ile Ile Phe Ser Ala Val Val 1 5 10 15 SerAla Gly Arg Tyr Pro Glu Thr Thr Val Gly Asn Leu Thr Lys Ser 20 25 30 PheGln Ala Pro Arg Leu Asp Arg Ser Val Gln Ser Pro Ile Tyr Asn 35 40 45 IlePhe Thr Asn His Val Ala Gly Tyr Ser Leu Ser His Ser Leu Tyr 50 55 60 AspArg Ile Val Phe Leu Cys Thr Ser Ser Ser Asn Pro Val Asn Gly 65 70 75 80Ala Cys Pro Thr Ile Gly Thr Ser Gly Val Gln Tyr Gly Thr Thr Thr 85 90 95Ile Thr Leu Gln Phe Thr Glu Lys Arg Ser Leu Ile Lys Arg Asn Ile 100 105110 Asn Ile Ala Gly Asn Lys Lys Pro Ile Trp Glu Asn Gln Ser Cys Asp 115120 125 Phe Ser Asn Ile Met Val Leu Asn Ser Lys Ser Trp Ser Cys Gly Ala130 135 140 His Gly Asn Ala Asn Gly Thr Ile Leu Asn Leu Tyr Ile Pro AlaGly 145 150 155 160 Glu Ile Asn Lys Leu Pro Phe Gly Gly Ile Trp Glu AlaThr Leu Ile 165 170 175 Leu Arg Leu Ser Arg Tyr Gly Glu Val Ser Ser ThrHis Tyr Gly Asn 180 185 190 Tyr Thr Val Asn Ile Thr Val Asp Leu Thr AspLys Gly Asn Ile Gln 195 200 205 Val Trp Leu Pro Gly Phe His Ser Asn ProArg Val Asp Leu Asn Leu 210 215 220 Arg Pro Ile Gly Asn Tyr Lys Tyr SerGly Ser Asn Ser Leu Asp Met 225 230 235 240 Cys Phe Tyr Asp Gly Tyr SerThr Asn Ser Asp Ser Met Val Ile Lys 245 250 255 Phe Gln Asp Asp Asn ProThr Asn Ser Ser Glu Tyr Asn Leu Tyr Lys 260 265 270 Ile Gly Gly Thr GluLys Leu Pro Tyr Ala Val Ser Leu Ile Gly Glu 275 280 285 Lys Ile Phe TyrPro Val Asn Gly Gln Ser Phe Thr Ile Asn Asp Ser 290 295 300 Ser Val LeuGlu Thr Asn Trp Asn Arg Val Thr Ala Val Ala Met Pro 305 310 315 320 GluVal Asn Val Pro Val Leu Cys Trp Pro Ala Arg Leu Leu Leu Asn 325 330 335Ala Asp Val Asn Ala Pro Asp Ala Gly Gln Tyr Ser Gly Gln Ile Tyr 340 345350 Ile Thr Phe Thr Pro Ser Val Glu Asn Leu 355 360 <210> SEQ ID NO 32<211> LENGTH: 353 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: ETEC Protein Homology Sequence<400> SEQUENCE: 32 Met Ser Asn Ile Cys Lys Trp Thr Ser Met Thr Ala HisTrp Ser Ala 1 5 10 15 Ile Ile Asn Phe Ile Arg Lys Tyr Val Tyr Pro AlaArg Ile Ile Ala 20 25 30 Ile Leu Ala Gly Ala Thr Leu Pro Gln Val Ala AspAla Ile Thr Val 35 40 45 Asp Leu Asn Tyr Asp Lys Asn Asn Val Ala Val IleThr Pro Val Trp 50 55 60 Ser Gln Glu Trp Ser Val Ala Asn Val Leu Gly GlyTrp Val Cys Arg 65 70 75 80 Ser Asn Arg Asn Glu Asn Glu Gly Cys Glu GluThr His Leu Val Trp 85 90 95 Trp Tyr Ala Phe Gly Ala Tyr Ser Ile Arg LeuArg Phe Arg Glu Gln 100 105 110 Ile Ser His Ala Glu Ile Thr Leu Ile LeuLeu Gly Ser Val Arg Asp 115 120 125 Ala Cys Thr Gly Val Ile Asn Met AsnAla Ala Ala Cys Gln Trp Gly 130 135 140 Arg Ser Leu Lys Leu Arg Ile ProSer Glu Glu Leu Ala Lys Ile Pro 145 150 155 160 Thr Ser Gly Thr Trp LysAla Thr Leu Val Leu Asp Tyr Leu Gln Trp 165 170 175 Gly Gly Asp Asp ProLeu Gly Thr Ser Thr Thr Asp Ile Thr Leu Asn 180 185 190 Val Thr Asp HisPhe Ala Glu Asn Ala Ala Ile Tyr Phe Pro Gln Phe 195 200 205 Gly Thr AlaThr Pro Arg Val Asp Leu Asn Leu His Arg Met Asn Ala 210 215 220 Ser GlnMet Ser Gly Arg Ala Asn Leu Asp Met Cys Leu Tyr Asp Gly 225 230 235 240Gly Val Lys Ala Arg Ser Leu Gln Met Met Glu Gly Ser Asn Lys Ser 245 250255 Gly Thr Gly Phe Gln Val Ile Lys Ser Asp Ser Ala Asp Thr Ile Asp 260265 270 Tyr Ala Val Ser Met Asn Tyr Gly Gly Arg Ser Ile Pro Val Thr Arg275 280 285 Gly Val Glu Phe Ser Leu Asp Asn Val Asp Lys Ala Ala Thr ArgPro 290 295 300 Val Val Leu Pro Gly Gln Arg Gln Ala Val Arg Cys Val ProVal Pro 305 310 315 320 Leu Thr Leu Thr Thr Gln Pro Phe Asn Ile Arg GluLys Arg Ser Gly 325 330 335 Glu Tyr Gln Gly Thr Leu Thr Val Thr Met LeuMet Gly Thr Gln Thr 340 345 350 Pro <210> SEQ ID NO 33 <211> LENGTH: 165<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: ETEC Protein Homology Sequence <400> SEQUENCE: 33 MetLys Leu Lys Lys Thr Ile Gly Ala Met Ala Leu Thr Thr Met Phe 1 5 10 15Val Ala Met Ser Ala Ser Ala Val Glu Lys Asn Ile Thr Val Thr Ala 20 25 30Ser Val Asp Pro Thr Ile Asp Ile Leu Gln Ala Asp Gly Ser Ser Leu 35 40 45Pro Thr Ala Val Glu Leu Thr Tyr Ser Pro Ala Ala Ser Arg Phe Glu 50 55 60Asn Tyr Lys Ile Ala Thr Lys Val His Thr Asn Val Ile Asn Lys Asn 65 70 7580 Val Leu Val Lys Leu Val Asn Asp Pro Lys Leu Thr Asn Val Leu Asp 85 9095 Ser Thr Lys Gln Leu Pro Ile Thr Val Ser Tyr Gly Gly Lys Leu Ser 100105 110 Thr Ala Asp Val Thr Phe Glu Pro Ala Glu Leu Asn Phe Gly Thr Ser115 120 125 Gly Val Thr Gly Val Ser Ser Ser Gln Asp Leu Val Ile Gly AlaThr 130 135 140 Thr Ala Gln Ala Pro Ser Ala Asn Tyr Ser Gly Val Val SerIle Leu 145 150 155 160 Met Thr Leu Ala Ser 165 <210> SEQ ID NO 34 <211>LENGTH: 168 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: ETEC Protein Homology Sequence <400>SEQUENCE: 34 Met Lys Phe Lys Lys Thr Ile Gly Ala Met Ala Leu Thr Thr MetPhe 1 5 10 15 Val Ala Val Ser Ala Ser Ala Val Glu Lys Asn Ile Thr ValThr Ala 20 25 30 Ser Val Asp Pro Ala Ile Asp Leu Leu Gln Ala Asp Gly AsnAla Leu 35 40 45 Pro Ser Val Lys Leu Ala Tyr Ser Pro Ala Ser Lys Ile PheGlu Ser 50 55 60 Tyr Arg Val Met Thr Gln Val His Thr Asn Asp Ala Thr LysLys Val 65 70 75 80 Ile Val Lys Leu Ala Asp Thr Pro Gln Leu Thr Asp ValLeu Asn Ser 85 90 95 Thr Val Gln Met Pro Ile Ser Val Ser Trp Gly Gly ValLeu Ser Thr 100 105 110 Thr Ala Lys Glu Phe Glu Ala Ala Ala Leu Gly TyrSer Ala Ser Gly 115 120 125 Val Asn Gly Val Ser Ser Ser Gln Glu Leu ValIle Ser Ala Ala Pro 130 135 140 Lys Thr Ala Gly Thr Ala Pro Thr Ala GlyAsn Tyr Ser Gly Val Val 145 150 155 160 Ser Leu Val Met Thr Leu Gly Ser165 <210> SEQ ID NO 35 <211> LENGTH: 170 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ETEC ProteinHomology Sequence <400> SEQUENCE: 35 Met Lys Leu Lys Lys Thr Ile Gly AlaMet Ala Leu Ala Thr Leu Phe 1 5 10 15 Ala Thr Met Gly Ala Ser Ala ValGlu Lys Thr Ile Ser Val Thr Ala 20 25 30 Ser Val Asp Pro Thr Val Asp LeuLeu Gln Ser Asp Gly Ser Ala Leu 35 40 45 Pro Asn Val Ala Leu Thr Tyr SerPro Ala Val Asn Asn Phe Glu Ala 50 55 60 His Thr Ile Asn Thr Val Val HisThr Asn Asp Ser Asp Lys Gly Val 65 70 75 80 Val Val Lys Leu Ser Ala AspPro Val Leu Ser Asn Val Leu Asn Pro 85 90 95 Thr Leu Gln Ile Pro Val SerVal Asn Phe Ala Gly Lys Pro Leu Ser 100 105 110 Thr Thr Gly Ile Thr IleAsp Ser Asn Asp Leu Asn Phe Ala Ser Ser 115 120 125 Gly Val Asn Tyr ValSer Ser Thr Gln Lys Leu Ser Ile His Ala Asp 130 135 140 Ala Thr Arg ValThr Gly Gly Ala Leu Thr Ala Gly Gln Tyr Gln Gly 145 150 155 160 Leu ValSer Ile Ile Leu Thr Lys Ser Thr 165 170 <210> SEQ ID NO 36 <211> LENGTH:170 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: ETEC Protein Homology Sequence <400> SEQUENCE:36 Met Lys Leu Asn Lys Ile Ile Gly Ala Leu Val Leu Ser Ser Thr Phe 1 510 15 Val Ser Met Gly Ala Ser Ala Ala Glu Lys Asn Ile Thr Val Thr Ala 2025 30 Ser Val Asp Pro Thr Ile Asp Leu Met Gln Ser Asp Gly Thr Ala Leu 3540 45 Pro Ser Ala Val Asn Ile Ala Tyr Leu Pro Gly Glu Lys Arg Phe Glu 5055 60 Ser Ala Arg Ile Asn Thr Gln Val His Thr Asn Asn Lys Thr Lys Gly 6570 75 80 Ile Gln Ile Lys Leu Thr Asn Asp Asn Val Val Met Thr Asn Leu Ser85 90 95 Asp Pro Ser Lys Thr Ile Pro Leu Glu Val Ser Phe Ala Gly Thr Lys100 105 110 Leu Ser Thr Ala Ala Thr Ser Ile Thr Ala Asp Gln Leu Asn PheGly 115 120 125 Ala Ala Gly Val Glu Thr Val Ser Ala Thr Lys Glu Leu ValIle Asn 130 135 140 Ala Gly Ser Thr Gln Gln Thr Asn Ile Val Ala Gly AsnTyr Gln Gly 145 150 155 160 Leu Val Ser Ile Val Leu Thr Gln Glu Pro 165170 <210> SEQ ID NO 37 <211> LENGTH: 168 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ETEC ProteinHomology Sequence <400> SEQUENCE: 37 Met Lys Leu Lys Tyr Thr Ile Gly AlaMet Ala Leu Ser Thr Ile Phe 1 5 10 15 Val Ala Val Ser Ala Ser Ala ValGlu Lys Asn Ile Thr Val Thr Ala 20 25 30 Ser Val Asp Pro Thr Ile Asp IleLeu Gln Ala Asn Gly Ser Ala Leu 35 40 45 Pro Thr Ala Val Asp Leu Thr TyrLeu Pro Gly Ala Lys Thr Phe Glu 50 55 60 Asn Tyr Ser Val Leu Thr Gln IleTyr Thr Asn Asp Pro Ser Lys Gly 65 70 75 80 Leu Asp Val Arg Leu Val AspThr Pro Lys Leu Thr Asn Ile Leu Gln 85 90 95 Pro Thr Ser Thr Ile Pro LeuThr Val Ser Trp Ala Gly Arg Thr Leu 100 105 110 Ser Thr Ser Ala Gln LysIle Ala Val Gly Asp Leu Gly Phe Gly Ser 115 120 125 Thr Gly Thr Ala GlyVal Ser Asn Ser Lys Glu Leu Val Ile Gly Ala 130 135 140 Thr Thr Ser GlyLys Pro Ser Ala Gly Lys Tyr Gln Gly Val Val Ser 145 150 155 160 Ile ValMet Thr Gln Ser Thr Asn 165 <210> SEQ ID NO 38 <211> LENGTH: 142 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: ETEC Protein Homology Sequence <400> SEQUENCE: 38 Val AspPro Thr Ile Asp Ile Leu Gln Ala Asn Gly Ser Ala Leu Pro 1 5 10 15 ThrAla Val Asp Leu Thr Tyr Leu Pro Gly Ala Lys Thr Phe Glu Asn 20 25 30 TyrSer Val Leu Thr Gln Ile Tyr Thr Asn Asp Pro Ser Lys Gly Leu 35 40 45 AspVal Arg Leu Val Asp Thr Pro Lys Leu Thr Asn Ile Leu Gln Pro 50 55 60 ThrSer Thr Ile Pro Leu Thr Val Ser Trp Ala Gly Lys Thr Leu Ser 65 70 75 80Thr Ser Ala Gln Lys Ile Ala Val Gly Asp Leu Gly Phe Gly Ser Thr 85 90 95Gly Thr Ala Gly Val Ser Asn Ser Lys Glu Leu Val Ile Gly Ala Thr 100 105110 Thr Ser Gly Thr Ala Pro Ser Ala Gly Lys Tyr Gln Gly Val Val Ser 115120 125 Ile Val Met Thr Gln Ser Thr Asp Thr Ala Ala Pro Val Pro 130 135140 <210> SEQ ID NO 39 <211> LENGTH: 133 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ETEC ProteinHomology Sequence <400> SEQUENCE: 39 Val Asp Pro Lys Leu Asp Leu Leu GlnAla Asp Gly Thr Ser Leu Pro 1 5 10 15 Asp Ser Ile Ala Leu Thr Tyr SerSer Ala Ser Asn Asn Phe Glu Val 20 25 30 Tyr Ser Leu Asn Thr Ala Ile HisThr Asn Asp Lys Thr Lys Ala Val 35 40 45 Val Val Lys Leu Ser Ala Pro AlaVal Leu Ser Asn Ile Met Lys Pro 50 55 60 Ser Ser Gln Ile Pro Met Lys ValThr Leu Gly Gly Lys Thr Leu Ser 65 70 75 80 Thr Ala Asp Ala Glu Phe AlaAla Asp Thr Leu Asn Phe Gly Ala Ser 85 90 95 Gly Val Glu Asn Val Ser SerVal Gln Gln Leu Thr Ile His Ala Glu 100 105 110 Ala Ala Pro Pro Glu AlaGly Asn Tyr Gln Gly Val Ile Ser Leu Ile 115 120 125 Met Thr Gln Lys Thr130 <210> SEQ ID NO 40 <211> LENGTH: 134 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ETEC ProteinHomology Sequence <400> SEQUENCE: 40 Val Asp Pro Lys Leu Asp Leu Leu GlnAla Asp Gly Thr Ser Leu Pro 1 5 10 15 Asp Ser Ile Ala Leu Thr Tyr SerSer Ala Ser Asn Asn Phe Glu Val 20 25 30 Tyr Ser Leu Asn Thr Ala Ile HisThr Asn Asp Lys Ser Lys Gly Val 35 40 45 Val Val Lys Leu Ser Ala Ser ProVal Leu Ser Asn Ile Met Pro Asn 50 55 60 Ser Gln Ile Pro Met Lys Val ThrLeu Gly Gly Glu Thr Leu Asn Thr 65 70 75 80 Thr Asp Thr Glu Phe Thr ValAsp Thr Leu Asn Phe Gly Thr Ser Gly 85 90 95 Val Glu Asn Val Ser Ser ThrGln Gln Leu Thr Ile His Ala Asp Thr 100 105 110 Gln Gly Thr Ala Pro GluAla Gly Asn Tyr Gln Gly Ile Ile Ser Leu 115 120 125 Ile Met Thr Gln LysThr 130

What is claimed is:
 1. An immunogenic composition comprising: arecombinant product of a csa operon and a carrier.
 2. The immunogeniccomposition of claim 1, wherein the recombinant product of the csaoperon is CsaA (SEQ ID NO.:2).
 3. The immunogenic composition of claim1, wherein the recombinant product of the csa operon is at least 95%homologous to CsaA (SEQ ID NO.:2).
 4. The immunogenic composition ofclaim 1, wherein the recombinant product of the csa operon is CsaB (SEQID NO.:4).
 5. The immunogenic composition of claim 1, wherein therecombinant product of the csa operon is at least 95% homologous to CsaB(SEQ ID NO.:4).
 6. The immunogenic composition of claim 1, wherein therecombinant product of the csa operon is CsaC (SEQ ID NO.:6).
 7. Theimmunogenic composition of claim 1, wherein the recombinant product ofthe csa operon is at least 95% homologous to CsaC (SEQ ID NO.:6).
 8. Theimmunogenic composition of claim 1, wherein the recombinant product ofthe csa operon is CsaD (SEQ ID NO.:8).
 9. The immunogenic composition ofclaim 1, wherein the recombinant product of the csa operon is at least95% homologous to CsaD (SEQ ID NO.:8).
 10. The immunogenic compositionof claim 1, wherein the recombinant product of the csa operon is CsaE(SEQ ID NO.:10).
 11. The immunogenic composition of claim 1, wherein therecombinant product of the csa operon is at least 95% homologous to CsaE(SEQ ID NO.:10).
 12. The immunogenic composition of claim 1, wherein thecarrier is a composition comprising components suitable for parenteraladministration.
 13. The immunogenic composition of claim 12, wherein thecarrier is a composition comprising components suitable for intranasaladministration.
 14. The immunogenic composition of claim 12, wherein thecarrier is a composition comprising components suitable forintramuscular administration.
 15. The immunogenic composition of claim1, wherein the carrier is a composition comprising components suitablefor enteric administration.
 16. The immunogenic composition of claim 1,wherein the recombinant product of the csa operon is an expressionvector comprising the csa operon or a fragment thereof.
 17. An isolatednucleotide sequence comprising a csa operon or a functional fragmentthereof.
 18. The isolated nucleotide sequence of claim 17, wherein thecsa operon comprises a csaA coding region.
 19. The isolated nucleotidesequence of claim 18, wherein the csa operon comprises the csaA codingregion of SEQ ID NO:
 1. 20. The isolated nucleotide sequence of claim18, wherein the csa operon comprises a nucleotide sequence having atleast 95% sequence homology to the csaA coding region of SEQ ID NO: 1.21. The isolated nucleotide sequence of claim 17, wherein the csa operoncomprises csaB.
 22. The isolated nucleotide sequence of claim 21,wherein the csa operon comprises the csaB coding region of SEQ ID NO: 3.23. The isolated nucleotide sequence of claim 21, wherein the csa operoncomprises a nucleotide sequence having at least 95% sequence homology tothe csaB coding region of SEQ ID NO:
 3. 24. The isolated nucleotidesequence of claim 17, wherein the csa operon comprises csaC.
 25. Theisolated nucleotide sequence of claim 24, wherein the csa operoncomprises the csaC coding region of SEQ ID NO:
 5. 26. The isolatednucleotide sequence of claim 24, wherein the csa operon comprises anucleotide sequence having at least 95% sequence homology to the csaCcoding region of SEQ ID NO:
 5. 27. The isolated nucleotide sequence ofclaim 17, wherein the csa operon comprises csaD.
 28. The isolatednucleotide sequence of claim 24, wherein the csa operon comprises thecsaD coding region of SEQ ID NO:
 7. 29. The isolated nucleotide sequenceof claim 24, wherein the csa operon comprises a nucleotide sequencehaving at least 95% sequence homology to the csaD coding region of SEQID NO:
 7. 30. The isolated nucleotide sequence of claim 17, wherein thecsa operon comprises csaE.
 31. The isolated nucleotide sequence of claim30, wherein the csa operon comprises the csaE coding region of SEQ IDNO:
 9. 32. The isolated nucleotide sequence of claim 30, wherein the csaoperon comprises a nucleotide sequence having at least 95% sequencehomology to the csaE coding region of SEQ ID NO:
 9. 33. An expressionvector comprising a csa operon nucleotide sequence or an antigenicfragment thereof.
 34. A host cell comprising the expression vector ofclaim
 33. 35. A purified polypeptide sequence expressed from arecombinant csa operon or an antigenic fragment thereof.
 36. Thepurified polypeptide sequence of claim 35, wherein the csa operoncomprises a csaA coding region.
 37. The purified polypeptide sequence ofclaim 35, wherein the csa operon comprises the csaA coding region of SEQID NO:
 1. 38. The purified polypeptide sequence of claim 37, wherein thecsa operon comprises a nucleotide sequence having at least 95% sequencehomology to the csaA coding region of SEQ ID NO:
 1. 39. The purifiedpolypeptide sequence of claim 35, wherein the csa operon comprises csaB.40. The purified polypeptide sequence of claim 39, wherein the csaoperon comprises the csaB coding region of SEQ ID NO:
 3. 41. Thepurified polypeptide sequence of claim 39, wherein the csa operoncomprises a nucleotide sequence having at least 95% sequence homology tothe csaB coding region of SEQ ID NO:
 3. 42. The purified polypeptidesequence of claim 35, wherein the csa operon comprises csaC.
 43. Thepurified polypeptide sequence of claim 42, wherein the csa operoncomprises the csaC coding region of SEQ ID NO:
 5. 44. The purifiedpolypeptide sequence of claim 42, wherein the csa operon comprises anucleotide sequence having at least 95% sequence homology to the csaCcoding region of SEQ ID NO:
 5. 45. The purified polypeptide sequence ofclaim 35, wherein the csa operon comprises csaD.
 46. The purifiedpolypeptide sequence of claim 45, wherein the csa operon comprises thecsaD coding region of SEQ ID NO:
 7. 47. The purified polypeptidesequence of claim 45, wherein the csa operon comprises a nucleotidesequence having at least 95% sequence homology to the csaD coding regionof SEQ ID NO:
 7. 48. The purified polypeptide sequence of claim 35,wherein the csa operon comprises csaE.
 49. The purified polypeptidesequence of claim 48, wherein the csa operon comprises the csaE codingregion of SEQ ID NO:
 9. 50. The purified polypeptide sequence of claim48, wherein the csa operon comprises a nucleotide sequence having atleast 95% sequence homology to the csaE coding region of SEQ ID NO: 9.51. A method of generating an immune response, comprising: providing animmunogenic composition to a subject, wherein said immunogeniccomposition comprises a recombinant product of a csa operon; andcontacting said subject with said immunogenic composition, whereby animmune response is generated in said subject.
 52. The method of claim51, wherein the product of the csa operon is the CS4 antigen.
 53. Themethod of claim 52, wherein the CS4 antigen is provided in an acellularcomposition.
 54. The method of claim 52, wherein the CS4 antigen isprovided in a cellular composition.
 55. The method of claim 51, whereinthe recombinant product of the csa operon is CsaA (SEQ ID NO.:2). 56.The method of claim 51, wherein the recombinant product of the csaoperon is at least 95% homologous to CsaA (SEQ ID NO.:2).
 57. The methodof claim 51, wherein the recombinant product of the csa operon is CsaB(SEQ ID NO.:4).
 58. The method of claim 51, wherein the recombinantproduct of the csa operon is at least 95% homologous to CsaB (SEQ IDNO.:4).
 59. The method of claim 51, wherein the recombinant product ofthe csa operon is CsaC (SEQ ID NO.:6).
 60. The method of claim 51,wherein the recombinant product of the csa operon is at least 95%homologous to CsaC (SEQ ID NO.:6).
 61. The method of claim 51, whereinthe recombinant product of the csa operon is CsaD (SEQ ID NO.:8). 62.The method claim 51, wherein the recombinant product of the csa operonis at least 95% homologous to CsaD (SEQ ID NO.:8).
 63. The method ofclaim 51, wherein the recombinant product of the csa operon is CsaE (SEQID NO.:10).
 64. The method of claim 51, wherein the recombinant productof the csa operon is at least 95% homologous to CsaE (SEQ ID NO.:10).65. The method of claim 51, wherein the carrier is a compositioncomprising components suitable for parenteral administration.
 66. Themethod of claim 65, wherein the carrier is a composition comprisingcomponents suitable for intranasal administration.
 67. The method ofclaim 65, wherein the carrier is a composition comprising componentssuitable for intramuscular administration.
 68. The method of claim 51,wherein the carrier is a composition comprising components suitable forenteric administration.
 69. A method of producing a polypeptide productfrom a csa operon or functional fragment thereof, comprising: providingthe csa operon in an expression vector; introducing the expressionvector into a host cell, such that a recombinant host cell is produced;and subjecting to the recombinant host cell to conditions such that aprotein from the csa operon is expressed.
 70. The method of claim 69,wherein the polypeptide product of the csa operon is the CS4 antigen.71. The method of claim 69, wherein the polypeptide product of the csaoperon is CsaA (SEQ ID NO.:2).
 72. The method of claim 69, wherein thepolypeptide product of the csa operon is at least 95% homologous to CsaA(SEQ ID NO.:2).
 73. The method of claim 69, wherein the polypeptideproduct of the csa operon is CsaB (SEQ ID NO.:4).
 74. The method ofclaim 69, wherein the polypeptide product of the csa operon is at least95% homologous to CsaB (SEQ ID NO.:4).
 75. The method of claim 69,wherein the polypeptide product of the csa operon is CsaC (SEQ IDNO.:6).
 76. The method of claim 69, wherein the polypeptide product ofthe csa operon is at least 95% homologous to CsaC (SEQ ID NO.:6). 77.The method of claim 69, wherein the polypeptide product of the csaoperon is CsaD (SEQ ID NO.:8).
 78. The method claim 69, wherein thepolypeptide product of the csa operon is at least 95% homologous to CsaD(SEQ ID NO.:8).
 79. The method of claim 69, wherein the polypeptideproduct of the csa operon is CsaE (SEQ ID NO.:10).
 80. The method ofclaim 69, wherein the polypeptide product of the csa operon is at least95% homologous to CsaE (SEQ ID NO.:10).
 81. A method for generating animmune response in a vertebrate against ETEC, comprising administeringto the vertebrate an amount of a polynucleotide operatively encoding atleast an immunogenic portion of the csa operon and having at least about15 nucleotides, or administering a polypeptide encoded thereby.